LIBRARY 
CALIFORNIA 

COLLEGE 
)F    PHARMACY 


BA« 
DE 


MEMCAL 


COLLEfffi  OF  PHARMACY 


Oa!!farr,!a  Co!?or-s  of  Pharmacy 


THE  YEASTS 


THE    YEASTS 


BY 
ALEXANDRE  ^UILLIEEMOND,  D.So. 

PROFESSOR  OF  BOTANY,  UNIVERSITY  OF  LYON 


TRANSLATED  AND  THOROUGHLY  REVISED 

IN  COLLABORATION 
WITH  THE  ORIGINAL  AUTHOR 

BY 

FRED   WILBUR  TANNER,  M.S.,  PH.D. 

ASSISTANT  PROFESSOR  IN   BACTERIOLOGY,   UNIVERSITY  OF   ILLINOIS 


OaHfcrnla  Co!!eso  of  Pharmacy 


NEW  YORK 

JOHN    WILEY    AND    SONS,   INC. 

LONDON:    CHAPMAN  AND  HALL,   LIMITED 

1920 

^    - 


COPYRIGHT • IQ20 
BY    FRED    W.     TANNER 


THE-PLIMPTON-PRESS 
NORWOOD-MASS-U-S-A 


FOREWORD 

NO  class  of  microoiganisms  has  been  more  intimately  associated 
with  the  progress  and  development  of  the  human  race  than  the 
yeasts.  Since  the  earliest  times,  these  microorganisms  have 
been  used  to  bring  about  changes  which  it  would  have  been  difficult  to 
have  accomplished  by  other  methods.  Since  microscopic  examinations 
have  revealed  the  presence  of  yeast  cells  in  bread  found  with  Egyp- 
tian mummies,  it  is  known  that  these  people  were  familiar  with 
yeast  fermentations,  although  they  probably  did  not  have  explana- 
tions for  the  changes  which  were  observed.  The  Norsemen  prepared 
an  alcoholic  drink  from  milk,  as  is  done  today  by  certain  nomadic 
races,  the  fermentation  of  which  was,  in  part,  caused  by  yeasts.  To- 
day we  find  the  yeasts  of  ever-increasing  interest  and  importance. 
The  food  microbiologist  must  understand  the  physiology  of  these 
organisms  if  he  is  to  successfully  cope  with  them.  They  are  assuming 
greater  importance  in  medicine,  especially  in  relation  to  certain  de- 
ficiency diseases,  constipation,  and  skin  infections.  Great  industries 
have  been  established  which  rest  entirely  on  the  chemical  changes 
brought  about  by  yeasts  and  their  enzymes;  some  of  them  would  be 
developed  with  difficulty,  we.re  it  necessary  to  use  strictly  chemical 
methods.  The  compressed  yeast  industry  itself  has  reached  a  high 
state  of  development  with  its  several  factories  located  in  different 
parts  of  America  and  distributing  agencies  in  practically  all  of  the 
cities  and  villages.  Many  industries  have  been  greatly  changed  by 
the  availability  of  fresh,  active  yeast  whenever  it  is  needed.  Despite 
the  facts  that  yeasts  have  always  been  of  great  significance  to  the 
human  race  and  that  they  will  probably  have  greater  significance  in 
the  future,  it  remained  for  Guilliermond  to  collect  the  various  data, 
which  have  accumulated  in  regard  to  them,  into  one  volume.  Several 
treatises  have  been  prepared  which  deal  with  the  yeasts  in  relation  to 
fermentations,  but  no  real  definitive  treatise  on  the  yeasts,  as  such, 
has  appeared  which  is  comparable  to  the  volume  prepared  by  Guillier- 
mond. The  investigations  of  this  authority  make  the  book  especially 
valuable.  These  facts  made  it  seem  advisable  to  translate  the  vol- 
ume for  publication  in  the  English  language  in  order  that  the  data 
might  be  available  to  the  practitioners  and  students  who  do  not  read 

v 

'  42;]?;;, 


vi  FOREWORD 

the  French  language.  It  is  sincerely  hoped,  however,  that  the  rendition 
of  this  book  into  the  English  language  will  in  no  way  inhibit  the 
study  of  the  French  language  —  that  language  in  which  Pasteur, 
Bernard,  Magendie,  Berthelot,  and  others  have  published  their  classic 
investigations. 

This  English  edition  is  based  on  Guilliermond's  "  Les  Levures," 
which  was  published  in  1912,  appearing  as  a  volume  in  the  section  on 
Cryptogamic  Botany  of  Encyclopedic  Scientifique.  This  series  is  edited 
under  the  direction  of  Doctor  Toulouse.  To  merely  translate  a 
volume  on  a  subject  which  is  being  developed  as  rapidly  as  the 
yeasts  would  be  entirely  inadequate.  Consequently  with  the  collab- 
oration of  Professor  Guilliermond,  the  translator  has  added  much 
new  material  which  has  been  published  since  1912.  Without  the 
assistance  of  Professor  Guilliermond,  this  could  not  have  been  done 
as  completely.  The  English  edition  may  not,  then,  be  regarded  as  a 
mere  translation  of  the  last  French  edition. 

The  rendition  of  a  text  from  a  foreign  language  into  the  English 
language  is  beset  with  difficulties  which  are  most  clearly  appreciated 
by  those  who  have  performed  similar  pieces  of  work.  In  all  cases 
a  literal  translation  has  not  been  attempted;  however,  the  opinions 
of  the  original  author  have  been  given  as  closely  as  possible.  It  is 
trusted  that  this  English  edition  will  make  it  easier  for  students  to 
pursue  their  study  of  these  important  microorganisms  and  for  the 
practitioner  to  more  easily  solve  the  problems  with  which  he  has  to 
cope.  I  owe  many  thanks  to  my  colleagues,  too  numerous  to  men- 
tion here,  for  expression  of  their  advice  at  various  times  and  for 
their  interest  during  the  progress  of  the  work. 

UNIVERSITY  OF  ILLINOIS  FRED   W.    TANNER 

URBANA,  ILLINOIS 
June,  1919 


AUTHOR'S  PREFACE 

SINCE  the  celebrated  memoir  by  Pasteur  on  alcoholic  fermenta- 
tion, the  yeasts  have  never  ceased  to  assume  an  ever-increasing 
importance  in  agriculture  and  the  industries.  The  classic  investi- 
gations by  Pasteur,  followed  by  those  of  Hansen,  have  shown  the 
profit  that  may  result  from  a  methodical  study  of  the  various  species 
of  yeasts,  by  a  knowledge  of  the  conditions  necessary  for  their  develop- 
ment and  biochemical  characteristics  for  application  to  the  fermenta- 
tion industries.  No  one  may  overlook  the  benefits  which  came  to  such 
industries  by  the  use  of  pure  cultures  and  selected  species,  and  the 
avoidance  of  yeasts  which  caused  defects  in  fermented  products.  The 
fermentologists  have  also  benefited  greatly  by  these  methods.  Finally, 
the  relatively  recent  investigations  have  shown  the  relationship  of 
yeasts  to  certain  diseases  in  man  and  animals. 

From  a  purely  theoretical  point  of  view  the  yeasts,  on  account  of 
the  facility  with  which  they  allow  themselves  to  be  cultivated  in  artifi- 
cial media,  and  by  the  relatively  large  size  of  their  cells,  are  especially 
favorable  objects  for  experimentation  upon  which  very  important 
investigations  of  physiology,  cytology  and  sexuality  have  been  made. 
They  have  contributed  appreciably  to  the  progress  of  general  physiol- 
ogy and  biology. 

It  seemed  useful  to  me  to  collect  into  one  book  all  of  the  knowledge 
required  on  the  morphology,  physiology  and  taxonomy  group  of  fungi, 
and  to  arrange  it  in  such  a  manner  that  the  data  would  be  available 
for  biologists,  practitioners  in  industrial  work,  agriculturalists  and 
physicians.  That  is  what  I  attempted  to  accomplish  in  the  little 
volume  published  in  the  Encyclopedic  Scicntifique  under  the  editorial 
supervision  of  Dr.  Toulouse. 

Professor  Tanner,  of  the  University  of  Illinois,  undertook  the 
translation  of  this  book  into  the  English  language  in  order  to  render  it 
more  accessible  to  American  students  and  American  investigators. 
This  is  indeed  a  great  honor  to  me,  one  which  I  did  not  dream  of  when 
I  prepared  this  modest  work  a  few  years  ago.  I  am  very  happy  to 
have  this  indication  of  friendship  between  scientific  America  and 
France,  a  friendship  which  I  hope  may  become  stronger  and  stronger. 
One  sufficiently  understands  the  significance  of  a  scientific  alliance  of 

vii 


viii  PREFACE 

two  nations  which  by  their  individual  characteristics  supplement  each 
other.  France  claims  such  great  teachers  as  Lamarck,  Claude  Bernard, 
and  Pasteur,  true  pioneers  in  the  field  of  biology.  By  her  spirit  per- 
haps, too  traditionalistic  and  suppressed  by  old  administrative  ma- 
chinery, she  has  not  always  understood  fully  the  real  utility  of  her 
universities  and  given  to  her  scientists  the  necessary  means  for  carry- 
ing on  their  work.  On  the  other  hand,  America,  with  no  such  rich 
heritage  from  the  past,  has  built  up  modern  laboratories  with  a  new 
spirit  and  equipped  them  with  the  necessary  resources.  She  probably 
possesses  the  greatest  universities  in  the  world.  She  lays  claim  to  able 
investigators  and,  thanks  to  her  marvelous  scientific  organization  and 
to  her  numerous  investigators,  is  sure  to  gain  very  rapidly  a  foremost 
place  in  the  scientific  world.  If  the  American  savants  have  the  desire 
to  profit  by  the  discoveries  of  the  French,  their  elders,  France  has 
much  to  gain  by  imitating  America  in  her  practical  ideas,  her  spirit  of 
organization,  her  methods  of  work,  and  her  tremendous  activity. 

The  book  which  Professor  Tanner  has  undertaken  to  present  to  the 
public  cannot  be  regarded  as  a  simple  translation  of  my  work;  it  is  a 
new  edition  resulting  from  intimate  collaboration  of  translator  and 
author.  Microbiology  is  progressing  so  rapidly  that  the  French  edi- 
tion, now  six  years  old,  is  no  longer  abreast  with  recent  acquisitions  of 
the  science.  It  was  found  necessary  to  make  numerous  editions  and 
to  modify  certain  chapters  in  which  Professor  Tanner  and  myself  have 
shared  the  labor.  Professor  Tanner,  known  by  his  work  on  the  bio- 
chemistry of  bacteria,  has  undertaken  the  revision  of  the  Chapter  on 
Physiology  of  the  Yeasts  which  was  no  small  task,  for  since  the  dis- 
covery of  zymase  by  Buchner,  the  biochemical  investigations  on  yeasts 
have  followed  each  other  without  interruption  and  have  become  in- 
creasingly valuable.  As  for  myself,  I  have  borne  the  task  of  revising 
the  Chapters  on  Morphology,  Phylogeny  and  Description  of  Species, 
subjects  with  which  I  am  more  familiar.  Professor  Tanner  had,  then, 
a  preponderant  part  in  the  translation  of  this  new  edition  and  the 
book  has  certainly  gained  much  by  the  collaboration  of  a  physiologist 
so  well  qualified. 

ALEXANDRE  GUILLIERMOND 

LYON,  September  8,  1919 


CONTENTS 

PAGE 

FOREWORD v 

AUTHOR'S  PREFACE vii 

INTRODUCTION 1 

History  of  the  Study  of  Yeasts. . . '. 3 


CHAPTER  I 
MORPHOLOGY  AND  DEVELOPMENT  OP  THE  YEASTS 

1.  General  Characters  of  Yeasts;  the  Different  Phases  in  Their  Development.  5 

2.  Forms  of  Yeast  Cells 6 

3.  Mycelial  Formations 8 

4.  Cell  Division 9 

a.  Budding 9 

b.  Transverse  Division 10 

5.  Durable  Cells 12 

6.  Sporulation 13 

7.  Sexuality 15 

a.  Copulation  Preceding  the  Formation  of  the  Asc 15 

b.  Copulation  of  Ascospores  or  Parthenogamy 23 

c.  Retrogradation  of  Copulation  —  Parthenogenesis 25 

8.  Germination  of  Ascospores 29 

a.  Direct  Germination  of  Ascospores  in  Asc 35 


CHAPTER  II 
CYTOLOGY  OF  YEAST 

1.  General  Considerations;  Historical 37 

2.  The  Nucleus 38 

3.  The  Cytoplasm  and  Its  Different  Constituents 39 

a.  Metachromatic  Corpuscles « 39 

b.  Glycogen 43 

c.  Basophile  Granules 44 

d.  Fats 44 

4.  The  Membrane 45 

5.  Changes  in  the  Cell  during  Fermentation 46 

6.  Cytological  Phenomena  during  Vegetative  Multiplication 47 

7.  Cytological  Phenomena  of  Sporulation 48 

ix 


x  CONTENTS 

PAGE 

CHAPTER  III 
PHYSIOLOGY,  NUTRITION,  RESPIRATION,  AND  ALCOHOLIC  FERMENTATION 

1.  Chemical  Composition  of  Yeasts 53 

2.  General  Considerations  of  the  Enzymes  in  Yeasts 58 

3.  Proteolytic  Enzymes  in  Yeasts 59 

a.  Proteases 59 

b.  Nucleases  or  Enzymes  of  Nucleo-Proteins 60 

4.  Lipase 61 

5.  Carbohydrate  Enzymes 61 

a.  Polysaccharides 62 

b.  Trisaccharides 63 

c.  Disaccharides 63 

d.  Glucosides : 65 

6.  Oxidizing  and  Reducing  Enzymes 66 

7.  Toxins 67 

8.  Nutrition  of  Yeasts 68 

a.  Mineral  Elements 68 

b.  Nitrogenous  Substances 69  , 

c.  Hydrocarbon  Compounds 75 

9.  Respiration 80 

10.  General  Characteristics  of  Alcoholic  Fermentation 81 

a.  Conditions  Necessary  for  Its  Production 82 

b.  General  Occurrence  of  Alcoholic  Fermentation 84 

c.  Comparison  of  Intramolecular  Respiration  with  Alcoholic  Fermen- 

tation    84 

d.  Differences  in  the  Fermenting  Function  in  Different  Yeasts 85 

11.  Fermentable  Sugars 86 

12.  Formula  of  Alcoholic  Fermentation  and  Secondary  Products 88 

13.  Characteristics  and  Properties  of  Buchner's  Zymase 89 

14.  Mode  of  Action  of  Zymase 94 

15.  General  Theories  of  Alcoholic  Fermentation 99 

a.  Pasteur's  Theory 99 

b.  Wortmann  and  Delbriick's 100 

c.  Theory  which  Makes  Fermentation  a  Phase  of  Respiration 101 

16.  Autophagy  or  Autolysis  of  Yeasts 104 


CHAPTER  IV 
PHYSIOLOGY  (continued) 

1.  Habitat  of  Yeasts 106 

2.  Duration  of  Life  of  Yeasts. 107 

3.  Action  of  Physical  Agents  on  Yeasts 108 

a.  Temperature 108 

b.  Light 109 

c.  Moisture 109 

d.  Metals  and  Salts 109 

e.  Pressure Ill 

/.    Antiseptics Ill 


CONTENTS  xi 

PAGE 

Physiological  Conditions  of  Budding 112 

Particular  Types  of  Budding.     Scums  and  Rings.     Physiological  Condi- 
tions for  their  Production 112 

Physiological  Conditions  of  Sporulation 114 

Parasitism  of  Yeasts.     Pathenogenic  Properties.     Symbiosis 120 

a.  Parasitism 120 

b.  Pathenogenic  Properties  of  Yeasts 122 

c.  Symbiosis  of  Yeasts — 124 


CHAPTER  V 

ORIGIN  OF  THE  YEASTS;  THEIR  POSITION  IN  CLASSIFICATIONS  OF  THE  FUNGI 
AND  THEIR  SYSTEMATIC  RELATIONSHIPS 

1.  Historical 131 

a.   Experiments  on  the  Transformation  of  Molds  into  Yeasts 131 

6.    Studies  in  Life  Cycles  of  Yeasts  in  Nature 134 

c.    Morphological  and  Cytological  Studies  on  Yeasts 137 

2.  Phylogeny  of  the  Yeasts.     Their  Affinities  in  the  Group  of  Ascomycetes.  139 


CHAPTER  VI 

METHODS  OF  CULTURE   AND   ISOLATION    OF    YEASTS.      PROCEDURES    FOR 

OBSERVATION 

1.  Methods  of  Culture 147 

a.   Culture  media 148 

6.    Methods  for  Obtaining  Sporulation 150 

2.  Methods  of  Purification  and  Isolation  of  Yeasts 153 

a.   Physiological  Methods 154 

6.    Dilution  Methods  for  Separating  Yeasts 154 

1.  Hansen's  Method 155 

2.  Lindner's  Method 157 

3.  Determination  of  the  Number  of  Cells  in  a  Culture  and  a  Study  of  the  Power 

of  Multiplication  of  Yeasts 157 

4.  Methods  for  Studying  the  Yeasts 15$ 

a.   Observation  on  the  Development  in  Moist  Chambers 158 

6.    Methods  for  Examining  the  Cytology  of  Yeasts 158 

c.  Methods  for  Determining  the  Action  of  Yeasts  Towards  the  Carbo- 

hydrates   161 

d.  Determination  of  Efficiency  of  Yeasts 163 

5.  Preservation  of  Yeasts .  .                                                                 164 


CHAPTER  VII 
METHODS  FOR  THE  CHARACTERIZATION  AND  IDENTIFICATION  OF  YEASTS 

1.  Character  of  the  Vegetation  in  the  Sediment 167 

2.  Shapes  and  Dimensions  of  Cells 167 

3.  Optimum  Temperatures  and  Temperature  Limits  for  Budding 167 


xii  CONTENTS 

PAGE 

4.  Thermal  Death  Points  of  Species  of  Yeasts 168 

5.  Temperature  Limits  and  Optimum  Temperature  for  Spore  Formation . . .  168 

6.  Sexuality,  Morphological  Characters  of  the  Ascs  and  Ascospores,  Germina- 

tion of  Ascospores 170 

7.  Temperature  Conditions  for  Scum  Formation  and  Ring  Formation 171 

8.  Macroscopic  Appearances  of  Cultures  on  Solid  Media 173 

9.  Giant  Colonies • 174 

10.  Biochemical  Activities  of  Yeasts ' 174 

11.  Methods  for  Determination  of  Species  Torula,  Mycoderma  and  Patho- 

genic Yeasts 176 


CHAPTER  VIII 

VARIATION  OF  SPECIES 

1.  Morphological  Variations 178 

a.  Temporary  Variations.     Polymorphism 178 

b.  Permanent  Variations.     Polymorphism 179 

2.  Physiological  Variations 184 


CHAPTER  IX 
CLASSIFICATION  OF  THE  YEASTS 

1.  Family  of  Saccharomycetes ' 193 

a.  First  Group 193 

b.  Second  Group 193 

c.  Third  Group 194 

d.  Fourth  Group , 195 

e.  Fifth  Group 195 

2.  Family  of  Non-Saccharomycetes 196 


CHAPTER  X 
FAMILY  OF  NON-SACCHAROMYCETES 

First  Group 

GENUS  I.  —  Sohizosaccharomyces. 

Schizosaccharomyces  octosporus 197 

"  Pombe,  Lindner 199 

"  mellacei,  Jorgensen 200 

"  asporus,  Eykmann 202 

"  aphalarae  calthae,  Sulc 202 

"  Formosensis,  Nakayaza 203 

"  Sautawensis,  Nakayaza 204 

"  Nokkoensis,  Nakayaza 204 

"  chermetis  abietis,  Sulc 205 


CONTENTS  xil 

Second  Group 

GENUS  II.  —  Zygosaccharomyces.  PAGE 

Zygosaccharomyces  Barkeri  (Barker),  Saccardo-Sydow 206 

"                 priorianus,  Klocker 206 

"               javanicus,  de  Kruyff 207 

"                japonicus,  Saito 207 

"                soya?  (Saito),  Guilliermond 209 

"                lactis  a,  Dombrowski 210 

"                Bailii  (?)  (Lindner),  Guilliermond 211 

manshuricus,  Saito 212 

mellis  addi,  Richter 212 

"                Nadsonii,  Guilliermond. 213 

"                major,  Takahashi  and  Yukawa 215 

chevalieri,  Guilliermond 216 

salsus,  Takahashi  and  Yukawa 218 

Asporogenic  Species  of  Zygosaccharomyces 219 

Yeast  F,  Pearse  and  Barker. 219 

Yeast  G,  Pearse  and  Barker 220 

Zygosaccharomyces  bisporus,  Anderson 220 

GENUS  III.  —  Debaromyces,  Klocker. 

Debaromyces  globosus,  Klocker 221 

"           tyrocola,  Konokotin 222 

GENUS  IV.  —  Nadsonia. 

Nadsonia  fulvescens '. 222 

"        elongata,  Konokotin 223 

GENUS  V.  —  Schwanniomyces,  Klocker. 

Schwanniomyccs  occidentalis 224 

GENUS  VI.  —  Torulaspora. 

Torulaspora  delbrucki,  Lindner . 225 

Yeast  E  and  F  of  Rose 225 

Saccharomyces  lactis  7,  Dombrowski. 226 

Third  Group 
GENUS  VII.  —  Saccharomycodes,  Hansen. 

Saccharomyces,  ludwigii,  Hansen 227 

"             Behrensianus,  Behrens 229 

"             comesii,  Cavara 230 

GENUS  VIII.  —  Saccharomycopsis  (Schionning). 

Saccharomyces  guttulatus  (Robin),  Schionning 230 

GENUS  IX.  —  Saccharomyces,  Meyen. 
A.  First  Sub-Group. 

Saccharomyces  cerevisiae,  Hansen 231 

Will's  Yeasts 233 

Yeast  of  Saaz  and  Frohberg 233 

Saccharomyces  carlsbergensis,  Hansen 234 

monacensis,  Hansen 235 

"             Pastorianus,  Hansen. 237 

"             intermedius,  Hansen 238 

validus 240 

Logus  Yeast,  Van  Laer  and  Denamur 241 

Saccharomyces  ettipsoideus,  Hansen 241 


xiv  CONTENTS 

PAGE 

Yeasts  Related  to  Ellipsoideus 242 

Johannisberg  Yeast  1 243 

Johannisberg  Yeast  II 243 

Saccharomyces  vini,  Miintzii 243 

"            turbtdans,  Hansen 244 

"            willianus,  Saccardo 245 

"             bayanus,  Saccardo 246 

"            ilids,  Gronlund 246 

"             aquifolii,  Gronlund 246 

"             piriformis,  Marshall- Ward 247 

"             vordermannii,  Went  and  Prinzen-Geerligs 247 

sake,  Yabe 247 

"             cartilaginosus,  Lindner 248 

"             batatae,  Saito 248 

"             multisporus,  Jorgensen 248 

"             mali  risleri,  Kayser 249 

Cider  Yeasts  of  Pearse  and  Barker 249 

Saccharomyces  Tokyo,  Nakazawa 250 

"             Yeddo,  Nakayaza 251 

Saccharomyces  of  Shiro-Koji,  Saito .  .  * 251 

"             T  and  V  of  Ludwig  Rose 252 

B.  Second  Sub-Group. 

Saccharomyces  marxianus,  Hansen 252 

"             manshuricus,  Saito 253 

"             exiguus,  Rees-Hansen 254 

"             Zopfii,  Artari 254 

"             Coreanus,  Saito 255 

"                    "         Forma  Major,  Saito 256 

"             Jorgensii,  Lasche 256 

C.  Third  Sub-Group. 

Saccharomyces  rouxii,  Boutroux. 256 

Yeast  from  Pulque,  No.  2,  Guilliermond 257 

Saccharomyces  soja,  Takahashi  and  Yukawa 258 

"             Lindneri,  Guilliermond 260 

"             paradoxus,  Barschniskaia , 260 

"             Mangini,  Guilliermond 261 

"             chevalieri,  Guilliermond 262 

"             Etienne,  Potron 263 

D.  Fourth  Sub-Group. 

Saccharomyces  mali  Duclauxi,  Kayser 264 

"             unisporus,  Jorgensen 264 

E.  Fifth  Group. 

Saccharomyces  fragilis,  Jorgensen 264 

"  flava  lactis,  Krueger . 265 

"  acidi  lactici,  Grotenfelt 266 

"  lactis  a,  Dombrowski 266 

"  lactis  ft  Dombrowski 266 

.  Yeast  from  Koumys,  Schipin 267 

Saccharomyces  anamensis,  Will 268 

"  taette,  major  and  minor,  Olsen-Sopp 268 


CONTENTS  xv 

F.   Sixth  Sub-Group.  PAGE 

Saccharomyces  conglomerates,  Reess 269 

Hansenii,  Zopf 269 

'•             theobromae,  Preyer 269 

Yeast  from  Salt,  Hoye 270 

Saccharomyces  anginae,  Achalme  and  Troisier 270 

"             tumefaciens  (Curtis),  Basse 270 

"             granulatus,  Vuillemin  and  Legrain 271 

"             Blanchardii  (Blanchard),  Guiart 271 

"             minor,  Engel 272 

uvarum,  Beijerinck 272 

"             Lemonniere,  Sartory  and  Laneur 273 

GENUS  X.  —  Hansenia,  Lindner-Klocker. 

Hansenia  apiculata,  Lindner 273 

"         valbyensis,  Klocker 275 

Fourth  Group 
GENUS  XI.  —  Pichia. 

Pichia  membranaefaciens,  Hansen 275 

//  and  ///,  Pichii 277 

"      hyalospora,  Lindner 277 

"      taurica,  Siefert 278 

"      tamarindorum,  Siefert 278 

"      californica,  Siefert 278 

"      radaisii,  Lutz 279 

"      farinosa,  Lindner 279 

"      suavebleus,  Klocker 280 

"      alcoholophila,  Klocker 281 

"      polymorpha,  Klocker 281 

"      calliphora,  Klocker .  . 281 

"      mandshuricus,  Saito ". 282 

Yeast  from  Pulque  No.  1,  Guilliermond 282 

Pichia  orientalia,  Beijerinck 283 

Saccharomyces  mycoderma  punctisporus,  Melard 283 

GENUS  XII.  —  Willia,  Hansen 283 

Wittia  anomala,  Hansen 284 

Biological  Varieties  of  Willia  anomola ' . . . .  284 

Willia  anomola  7,  Steuber 285 

"      //,  Steuber 286 

"    ///,  Steuber 286 

"    IV,  Steuber 286 

11      belgica,  Lindner 287 

"      wichmanni,  Zikes 287 

"      anomala,  Saito 287 

"      salurnus,  Klocker 288 

Fifth  Group 

GENUS  XIII.  —  Monospora,  Metschnikoff. 

Monospora  cuspidatat  Metschnikoff 290 


xvi  CONTENTS 

GENUS  XIV.  —  Nematospora,  Peglion.  PAGE 

Nematospora  coryli,  Peglion 290 

"           Lycopersici,  Schneider 291 

GENUS  XV.  —  Coccidiascus,  Chatton 292 

Coccidiascus  Legeri,  Chatton 292 

CHAPTER  XI 
FAMILY  OF  NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

I.  GENUS  TORULA,  Turpin. 

A.  Torula  from  Breweries  or  Various  other  Sources. 

Hansen's  Torula 293 

Will's  Torula : 295 

Torula  of  Lindner  and  Meissner 299 

Torula  novae  cralsbergiae,  Gronlund 299 

"  brettanomyces,  Clausen 299 

Beijerinck's  Torula 300 

Torula  colliculosa,  Hartmann 301 

Saccharomyces  spec,  Saito 301 

"  awamori,  Inui 302 

De  Kruyff 's  Torula 302 

Torula  of  Pearse  and  Barker 302 

Saccharomyces  brassicae  I,  Wehmer 303 

"  II,  Wehmer 303 

///,  Wehmer 303 

Torula  holmii  (Holm),  Jorgensen 304 

"  thermantitonum,  Johnson 304 

Torula  from  Vaccine  Pulp,  Lesieur  and  Mangini 304 

Torula  of  Rose 305 

Wehmer's  Torula 305 

Torula  sp.,  Saito 306 

Hoyes  Torula 306 

B.  Torula  from  Milk. 

Torula  kephir,  Heinze  and  Cohn 307 

Saccharomyces  tyrocola,  Beijerinck 307 

Freudenreich's  Kephir  Yeast 307 

Duclaux's  Torula 307 

Torula  lactis  (Adanletz),  Heinze  and  Cohn , 308 

Kayser's  Yeast 309 

Torula  communis,  Browne 309 

Lactomyces  inflans  caseigana,  Bochicchio 310 

Saccharomyces  iebenis,  Rist  and  Khoury 310 

Torula  kephir,  Nokolajewa 311 

Torula  ellipsoidea,  Nikolajewa 311 

"      amara,  Harrison 311 

Dombrowski's  Torula 311 

C.  Yeasts  from  Fats. 

Saccharomyces  olei,  Van  Tieghem 313 

Roger's  Torula 313 


CONTENTS  xvii 

PAGE 

D.   Colored  Torula 314 

Torula  pulcherrima,  Lindner 314 

"      mucilaginosa,  Jorgensen 314 

"      dnnabarina,  Jorgensen 315 

Red  Torula,  No.  36,  Janssens  and  Martens 315 

Torvla  glutinis,  Pringsheim  and  Bilewsky 316 

Cryptococcus  bainieri,  Sartory 318 

Pseudosaccharomyces  Stevensi 318 

Cryptococcus  verrucosus,  Anderson 319 

ovoideus,  Anderson 320 

glabratus,  Anderson 320 

agregatus,  Anderson 321 

Kramer's  Red  Torula 321 

Saccharomyces  japonicus,  Yabe 322 

"             keiskeana,  Yabe 322 

Torula  'bogoriensis  rubra,  De  Kruyff 323 

"      rubefaciens,  Grosbusch 323 

GENUS  II.     Pseudosaccharomyces,  Klocker 323 

Pseudosaccharomyces  apiculatus,  Klocker. 323 

apiculatus  parasiticus,  Klocker 324 

Saccharomyces  macropsidis  lanionis,  Sulc 325 

Pseudosaccharomyces  austricus,  Klocker 325 

"                  Africanus,  Klocker 325 

"                  cortid,  Klocker 325 

"                  Mullen,  Klocker 325 

"                  Lindneri,  Klocker 326 

"                  Germanii,  Klocker 326 

Jensenii,  Klocker 326 

"                  Malaianus,  Klocker. . ; 326 

La/an,  Klocker 326 

Willii,  Klocker 326 

antillarum,  Klocker 327 

"                  occidentalis,  Klocker '. . : 327 

"           .       sautranzensis,  Klocker 327 

"                  indicus,  Klocker 327 

of  Will 327 

Torula  nigra,  Marpmann 328 

Torula  from  "Soya"  Mash,  Kita 330 

III.   GENUS  MYCODERMA,  Persoon. 

Mycoderma  cerevisiae,  Desm,  Hansen 331 

vini,  Desm 331 

Henneberg 333 

cucumerina,  Aderhold : . .  334 

"          valida,  Leberle-Will 334 

gallica,  Leberle-Will 334 

decolorans,  Wull 335 

Saito's  Mycoderma 335 

Brusendorf 's  Mycoderma 336 

Saccharomyces  mycoderma  I ,  Wehmer 336 

"                   "         II,       " 336 


xviii  CONTENTS 

PAGE 

DuClaux's  Yeast  (Mycolevure) 336 

Mycoderma  from  Pineapple,  Kayser 337 

"           lebenis,  Rist  and  Khoury 337 

Dombrowski's  Mycoderma  from  Milk 338 

Mycoderma  Chevalieri,  Guilliermond 339 

sp.,  Saito  . . . . : 340 

Mycoderma  of  Fischer  and  Brebeck 340 

"           monosa,  Anderson 341 

rugosa,  Anderson 341 

tannica,  Asai 342 

IV.   GENUS  MEDUSOMYCES,  Lindau. 

Medusomyces  Gisevii,  Lindau 343 


CHAPTER  XII 

PATHOGENIC  YEASTS 

Cryptococcus  degenerans,  Vuillemin 345 

gilchristi,  Vuillemin 345 

tokishigei  (Tokishige),  Vuillemin 346 

"  farciminosus,  Rivolta  and  Micellone 348 

"  hominis  (Busse),  Vuillemin 348 

"  linguae-pilosae,  Vuillemin 349 

"  Lithogenes,  Vuillemin 349 

Granulomatogenes,  Vuillemin 350 

niger  (Maffuci  and  Sirleo),  Vuillemin 350 

"  Plimmeri,  Costantin 351 

"  Corsellii,  Corselli  and  Frisco 351 

"  De  Gotti  and  Brazzola 351 

hominis  Costantini  (Constantin) 352 

Kleinii,  Erich  Cohn 352 

"  Anobii,  Escherich 352 

"  Parasitaris  (Trabut),  Vuillemin 353 

"  Psoriaris,  Rivolta 353 

"  Capillitii,  Vuillemin 353 

"  ovalis,  Vuillemin 353 

Cavicola,  Arthault 353 

neoformans,  San  Felice 354 

"  of  Clerc  and  Sartory 354 

Blastomyces  Hessleri,  Rettger 354 

Cryptococcus  ruber,  Vuillemin 355 

Guilliermondia,  Brauverie  and  Lesieur 355 

Lesieuri,  Beauverie  and  Lesieur 356 

sulfureus,  Beauverie  and  Lesieur 356 

rogeri,  Sartory  and  Demanche 356 

Salmoneus,  Sartory 357 

Le  Dantec's  Yeast 357 

Saccharomyces  membranogenes,  Steinhaus 357 

Atelosaccharomyces  of  Hudelo,  De  Beurmann  and  Gougerot 358 


CONTENTS  xix 

PAGE 

Atelosaccharomyces  of  Brewer  and  Wood 359 

Harteri  (Harter),  De  Beurmann  and  Gougerot 359 

Mercier's  Yeast 360 

Saccharomyces  Conomeli  and  Limbati,  Karel  Sulc 360 


CHAPTER  XIII 
FUNGI  RELATED  TO  THE  YEASTS 

Endomyces  albicans,  Vuillemin 361 

Parasaccharomyces  Ashfordii,  Anderson 364 

Thomasii,  Anderson 365 

Endomyces  Lindneri,  Saito 366 

"          Hordei,  Saito 366 

"          capsularis,  Guilliermond 367 

"         fibuliger,  Lindner 370 

javanensis,  Klocker 373 

"          Cruzi,  Mello  and  Paes 374 

Monilia  Candida,  Boborden 375 

Monilia  nigra,  Browne 378 

Monilia  fusca,  Browne 378 

Geiger's  Pseudomonilia 378 

Pseudomonilia  albomarginata,  Geiger 378 

rubescens,  Geiger 379 

"  mesentericus,  Geiger 379 

"  cartilaginosa,  Geiger 379 

Monilia  vini,  Osterwalder 379 

Parendomyces  pvlmonalis,  Plaut 380 

BIBLIOGRAPHICAL  INDEX 381 

INDEX  OF  NAMES 411 

INDEX 417 


THE   YEASTS 


THE  YEASTS 

INTRODUCTION 
What  are  Yeasts? 

"|"  TNDER  the  name  of  yeasts  have  been  generally  grouped  all  micro- 
«  organisms  which,  when  placed  in  sugar  solutions,  decompose 
V*^  them  into  alcohol  and  carbon  dioxide — cause  alcoholic  fermen- 
tation. Knowledge  with  regard  to  the  chemical  properties  of  the  yeasts 
has,  to  a  great  extent,  preceded  that  with  regard  to  their  nature. 
The  old  word  yeasts  (Fr.  levure  =  Latin  lever)  which  emphasized 
their  chemical  properties  dates  from  an  epoch  when  no  attention  was 
given  to  their  biological  significance  or  nature.  But  today  the 
name  yeast  has  taken  on  a  restricted  meaning  among  botanists. 
In  the  botanical  sense,  yeasts  are  unicellular  fungi  of  biochemical 
interest,  spherical  or  oval  in  shape,  and  which  multiply  by  budding.  A 
yeast,  then,  is  a  fungus  with  special  morphology.  Be  that  as  it  may,  the 
term  is  not  applied  to  an  indefinite  group  of  fungi  but  to  a  natural  one. 
Many  fungi,  more  or  less  developed,  living  normally  with  a  myce- 
lium are  able  to  reproduce  by  budding  of  their  filaments,  to  form 
cells  which  have  the  shapes  of  yeasts.  These  multiply  in  their  turn 
by  budding  and  retain  the  form  of  yeasts  for  many  generations. 
(Fig.  1.)  The  basidiospores  of  certain  Basidiomycetes  (Calcera  vis- 
cosa)  and  ascospores  of  certain  Ascomycetes  (Sphaerulina  intermixta 
Taphria)  give  rise  to  yeasts  and  it  is  only  after  living  for  a  certain 
time  in  this  form  that  the  yeast  cells  elongate  filaments  and  produce 
a  mycelium.  Among  the  Ustilaginaks,  the  sporidia,  which  spring 
from  the  promycelium,  exist  also  in  the  shape  of  yeasts;  it  is  this 
state  in  which  they  develop,  and  which  they  constantly  retain  when 
cultivated  in  artificial  media.  The  Mucors,  when  placed  in  sugar 
solutions,  are  able  to  dissociate  their  filaments  into  round  bodies,  or 
buds,  in  a  similar  manner  as  the  yeasts.  Dematium  pullulans  (Fig.  1), 
a  mold  with  a  well-differentiated  mycelium,  produces  in  a  regular 
fashion,  by  budding  of  its  filaments,  numerous  yeast  conidia;  when 
these  are  cultivated  under  certain  conditions,  they  are  transformed 
with  difficulty  into  mycelium.  Vegetation  with  forms  like  yeasts 
is,  then,  rather  widespread  among  the  fungi. 

Aside  from  these  fungi,  in  which  yeast  forms  are  merely  stages  of 
development,  there  are  others  which  live  constantly  in  the  forms  of 

1 


INTRODUCTION 


yeasts.  These  do  not  present  a  true  mycelium  at  any  time.  They 
reproduce  at  times  by  elongation  of  their  cells,  which  adhere 
together,  forming  structures  resembling  mycelium;  but  these  never 
offer  the  complexity  of  a  typical  mycelium.  In  the  category  of  yeasts 
belong  the  alcoholic  ferments  and  all  of  the  fungi  more  generally 
known  under  the  name  of  yeasts. 

These  yeasts,  which  are  often  designated  as  "  true  yeasts "  in 
contradistinction  to  "  yeast-like  fungi "  derived  from  more  highly 

developed  fungi,  are  not  distinguishable  in 
any  manner  from  the  latter.  The  general 
form  and  the  internal  characteristics  of  the 
cells  are  the  same  in  both  cases.  Physi- 
ologically, certain  true  yeasts  differ  only 
from  yeast  forms  of  molds  by  their  resist- 
ance to  anaerobic  conditions  and  excep- 
tional activity  of  the  fermenting  function, 
but  very  many  yeast-like  structures,  de- 
rived from  fungi  more  highly  developed, 
are  equally  capable  of  producing  alcoholic 
fermentation,  and  only  differ,  from  this 
point  of  view,  from  true  j^easts  by  a  de- 
creased activity  of  fermentation.  On  the 

F^l.-Dematiumpullulans.     °ther    hand>    a    certaif1    nUmbu6r    °f    tme 

1  to  4,  Mycelium  Forming  Yeasts;  5,    yeafits  ar6  totally  deprived  of  the  ferment- 

*>-ocess  of  ing  function.  It  is  understood  then,  how 
the  early  investigators  were  much  confused 
when  it  became  necessary  to  characterize  the  yeasts. 

In  the  meantime,  an  essential  difference  which  did  not  escape 
investigators  existed  between  the  yeast-like  fungi  and  the  yeasts 
properly  so-called.  Indeed,  most  of  the  true  yeasts  are  distinguished 
closely  from  "  yeast-forms  "  by  their  aptitude  to  produce  resistant 
endospores  at  certain  stages  in  their  life  cycles  (unfavorable  con- 
ditions), in  the  interior  of  their  cells;  the  cells  are  then  transformed 
into  sporangia.  De  Bary,  Rees,  and  Hansen  first  compared  these 
sporangia  to  ascs  of  Ascomycetes,  considering  the  true  yeasts  as  auton- 
omous fungi  which  live  only  in  the  form  of  yeasts  and  are  incapable 
of  developing  a  mycelium. 

This  conception  is  definitely  admitted  today,  as  we  shall  see 
when  the  origin  and  systematic  relationships  of  the  yeasts  are  taken  up. 
The  autonomy  of  the  yeasts  and  their  incorporation  as  a  group  of 
Ascomycetes  have  been  demonstrated  only  since  Hansen  observed  their 
life  cycles  in  nature  and  since  certain  investigators  have  given  evi- 
dence in  the  origin  of  the  asc  of  certain  yeasts,  of  the  presence  of 


Myc 

Yeast-Like    Bodies     m 
Budding  (after  Loew). 


INTRODUCTION  3 

a  sexuality  quite  comparable  to  that  which  is  observed  in  the  lower 
Ascomycetes.  The  yeasts  make  up  a  family  of  the  Ascomycetes 
known  as  Saccharomycetes. 

True  yeasts  never  produce  endo-  or  ascospores.  Do  they  represent 
forms  derived  from  more  highly  developed  fungi  and  made  constant 
by  a  long  adaptation  to  this  condition?  Or,  are  they  true  Saccharo- 
mycetes having  lost,  by  a  series  of  unknown  circumstances,  their  apti- 
tude of  forming  spores?  We  shall  see  that  a  definite  loss  of  this 
characteristic  has  often  been  proved  among  the  Saccharomycetes 
during  certain  special  conditions.  Be  that  as  it  may,  the  origin  of  the 
yeasts  is  entirely  ignored;  the  Saccharomycetes  are  then  separated 
and  regarded  as  yeasts  of  uncertain  origin.  In  this  book,  we  shall 
examine  extendedly  only  the  true  yeasts;  first,  yeasts  with  ascospores, 
or  Saccharomycetes;  secondly,  those  cells  which  exhibit  all  the 
characteristics  of  Saccharomycetes  with  the  exception  of  ascospore 
formation  and  which,  from  the  above,  one  might  call  pseudo-yeasts. 
All  of  those  yeast-like  structures  of  other  fungi  will  be  neglected. 
True  yeasts  are  very  abundant  in  nature;  over  five  hundred  are 
known.  The  limits  of  our  study  must  be  rather  wide. 

History  of  the  Study  of  Yeasts 

The  study  of  yeasts  is  intimately  associated  with  that  of  fermen- 
tation. The  idea  that  alcoholic  fermentations  are  caused  by  living 
organisms  originated  with  Linne.  In  1680  Leewenhoek  first  de- 
scribed the  yeasts.  He  described  them  as  globular  bodies,  oval  or 
spherical  in  shape.  In  1799  Fabroni  compared  yeasts  to  albu- 
minoids. About  1825,  and  for  some  time  after  this,  Mitscherlick, 
Cagnard-Latour,  Schwann,  and  Kiitzing  demonstrated  that  beer  and 
wine  yeasts  were  composed  of  cells  which  multiplied  by  budding. 
In  1839  Schwann  observed,  for  the  first  time,  endospores  in  yeasts. 
He  proved  that  they  might  be  freed  by  a  rupturing  of  the  cell  wall. 

But  the  nature  of  yeasts  has  been  definitely  known  since  the 
period  in  which  Pasteur  commenced  his  investigations  on  fermenta- 
tion. Up  to  this  time,  it  was  known  that  beer  yeast  multiplied 
when  introduced  into  saccharine  wort;  it  was  believed  that  it  was 
formed  spontaneously  and  that,  in  the  yeast,  was  an  occult  force 
which  produced  the  fermentation;  that  was  all  there  was  to  it. 
With  Pasteur,  definite  knowledge  with  regard  to  yeasts  commenced. 
It  was  in  1859  that  he  established,  by  his  memorable  experiments 
which  cannot  be  reproduced  here  on  account  of  the  lack  of  space, 
that  fermentation  is  correlative  with  the  life  of  yeasts.  Some  years 
later,  he  demonstrated  the  impossibility  of  spontaneous  generation 


4  INTRODUCTION 

and  introduced  the  methods  of  pure  culture  which  permitted  a  mor- 
phological study  of  yeasts. 

After  this,  the  yeasts  were  subjected  to  many  investigations.  In 
the  meantime,  the  methods  of  culture  were  not  quickly  taken  up, 
and  their  perfection  was  rather  slow.  It  was  also  difficult  for  the 
earlier  investigators  to  distinguish  between  yeasts  and  other  microor- 
ganisms which  developed  at  the  same  temperature.  The  early  work 
on  yeasts  conflicted  with  the  erroneous  conceptions  of  the  pleomor- 
phists  who  maintained  that  the  microorganisms  could  be  reduced  to 
a  small  number  of  species  capable  of  exhibiting  different  shapes  de- 
pendent upon  the  conditions.  About  1871,  Bechamp  reported  that 
the  acetic  acid  bacteria  could  change  into  yeasts.  In  1872,  Trecul 
thought  that  he  had  obtained  the  transformation  of  the  spores  of 
Penicillium  glaucum  into  yeasts.  In  187£,  Robin  stated  that  many  of 
the  yeasts  (Torula  cerevisiae,  Mycoderma  cerevisiae)  are,  with  Peni- 
cillium glaucum,  only  forms  of  the  same  fungus.  A  little  later,  how- 
ever, very  careful  investigations  were  reported.  Between  1868  and 
1870,  Rees  observed  endospores  in  many  species  of  yeasts,  and  gave 
the  first  accurate  description  of  these  organs.  This  was  followed  by 
the  work  of  Engel,  Seynes,  Brefeld,  and  de  Bary.  The  last  of  these, 
in  his  "  Morphology  and  Biology  of  the  Fungi,"  classed  the  yeasts 
among  the  Ascomycetes. 

The  introduction  and  perfection  of  pure  cultures  permitted  the 
exact  morphological  studies  of  the  yeasts.  This  was  the  work  of 
Hansen,  who  is  the  true  founder  of  this  study,  and  whose  name 
marks  a  second  step  in  the  history  of  the  yeasts.  Through  his  careful 
investigations  for  a  period  of  30  years,  this  mycologist  perfected 
methods  which  were  introduced  by  Pasteur  for  culturing  and  isolat- 
ing the  yeasts.  He  succeeded  in  inoculating  cultures  with  a  single  cell 
and  separating  one  species  from  another.  By  careful  studies  on  the 
morphological  and  physiological  properties  of  yeasts,  Hansen  found 
the  characteristics  which  allowed  the  differentiation  of  one  species 
from  another.  He  has  thus  been  able  to  characterize  a  large  number 
of  species  the  majority  of  which  are  known.  Hansen  is  responsible 
for  our  knowledge  of  the  life  cycle  and  the  systematic  relationships 
of  the  yeasts.  In  recent  years,  he  has  proposed  a  classification  which 
has  been  universally  accepted. 

The  third  step  in  the  study  of  yeasts  was  the  discovery,  by  Buch- 
ner,  of  zymase,  which  allowed  a  considerable  advance  in  the  study  of 
yeast  nutrition  and  the  mechanism  of  alcoholic  fermentation.  Thus, 
as  has  been  said,  three  names,  Pasteur,  Hansen  and  Buchner,  remain 
intimately  associated  with  the  study  of  the  yeasts  and  will  con- 
stitute the  pivot  about  which  our  investigations  will  center. 


PART  I  — GENERAL 

CHAPTER   I 
MORPHOLOGY  AND   DEVELOPMENT  OF  YEASTS 

General  Characteristics  of  Yeasts.   The  Different  Phases  in 
Their  Development 

THE  yeasts  are  unicellular  fungi;  generally,  they  live  in  isolated 
states.  After  they  have  acquired  a  certain  size,  they  divide  and 
produce  daughter  cells  which  are  not  slow  to  separate,  enlarge, 
and  divide  in  their  turn  in  the  same  manner. 

In  almost  all  of  the  yeasts,  cellular  division  takes  place  by  budding 
or  gemmation.  There  are  a  few  species  from  warm  climates  in  which 
multiplication  is  effected  by  transverse  division.  By  reason  of  this 
particular  method  of  division,  these  species  have  been  brought  together 
into  a  special  group  and  named  Schizosaccharomycetaceae. 

Budding  consists  in  the  appearance,  on  any  side  of  the  cell,  of  a 
little  bud  which  slowly  increases  in  size  until  it  ultimately  becomes  a  cell 
identical  with  the  mother  cell. 

Partition  simply  consists  of  the  formation  in  the  middle  of  the  cell 
of  a  wall  which  divides  the  cell  into  two  daughter  cells  of  equal  size 
which  grow  eventually. 

When  division  of  the  cell  takes  place  rapidly,  it  often  happens  that 
many  buds  are  formed  simultaneously  at  different  points  on  the  sur- 
face, and  these  daughter  cells  begin  to  multiply  before  separating. 
This  forms  a  little  colony,  or  conglomeration,  of  cells.  The  same  phe- 
nomenon is  observed  in  the  case  of  the  Schizosaccharomycetaceae. 

In  old  cultures  and  under  certain  conditions,  the  cells  remain  united 
in  long  chains;  this  gives  the  appearance  of  a  mycelium,  but  it  always 
remains  in  a  rudimentary  state. 

The  yeasts  are  then  able  to  present  two  forms:  one,  which  is  most 
frequent,  represents  the  normal  type  of  vegetation.  In  this  the 
cells  are  isolated  or  united  in  little  groups;  the  other,  which  is  quite 
exceptional,  is  the  filamentous  or  mycelial  state. 

When  the  yeast  finds,  in  the  medium  in  which  it  is  cultivated,  favor- 
able conditions  for  growth,  it  divides  actively  until  the  conditions 
become  unfavorable  from  the  lack  of  food  or  the  accumulation  of 

5 


6        MORPHOLOGY  AND  DEVELOPMENT  OF  PLANTS 

products  of  metabolism;  it  ceases,  then,  to  divide  and  produces  or- 
ganisms which  allow  it  to  perpetuate  itself  over  unfavorable  con- 
ditions. 

Will  and  Casagrandi,  under  these  conditions,  have  observed  cells 
iilled  with  reserve  products  (fats  and  glycogen)  enclosed  in  a  thick 
wall.  They  offer  a  great  resistance  thanks  to  these  reserve  products, 
which  they  retain  for  a  long  time  during  suspended  activity,  until 
favorable  conditions  allow  them  to  develop  again.  The  cells,  which 
are  comparable  to  cysts,  have  been  designated  under  the  name  of 
"  durable  cells." 

But  the  ordinary  process  employed  by  the  yeasts  for  perpetuation 
of  the  species  is  sporulation;  a  certain  number  of  internal,  or  endo- 
spores,  are  formed  in  the  interior  of  each  cell.  The  cells  are  thus  trans- 
formed into  a  sort  of  sporangium  which  is  called  an  asc.  The  spores  or 
ascospores  formed  in  these  ascs  are  endowed  with  a  great  resistance 
against  external  conditions  and  during  years  of  suspended  activity. 
When  placed  in  favorable  conditions,  they  swell  up  and  rupture  the 
asc  wall  to  become  free.  They  then  offer  the  appearance  of  vegeta- 
tive cells  and  multiply  in  the  ordinary  way. 

In  certain  species,  the  formation  of  the  asc  is  preceded  by  a  sexual 
process;  the  asc  then  results  from  the  fusion  of  two  cells  —  a  copulation 
as  in  the  case  of  an  egg.  In  other  species,  sexuality  is  maintained  in  a 
lower  state  of  development;  in  this  case,  it  takes  place  between  two 
spores  at  the  moment  of  germination.  In  the  greater  number  of  yeasts, 
however,  no  sexuality  has  been  observed. 

Many  of  the  yeasts,  as  Torula  and  Mycoderma,  do  not  form  endo- 
spores.  We  shall  investigate  successively,  in  this  chapter,  the  mor- 
phological characteristics  of  yeasts:  the  form  and  shape  of  the  cells, 
mycelial  formations,  durable  cells,  cellular  division,  formation  of  the 
asc,  sexuality,  and  germination  of  ascospores. 

Forms  of  Cells 

The  yeasts  offer  forms  varying  usually  from  a  sphere  to  an  ellipse. 
They  possess  quite  a  thick  membrane.  The  greater  number  of 
them  have  a  colorless  interior  containing  vacuoles  and  refractive 
granules.  Often,  a  red  pigment  may  be  observed,  sometimes  a  brown, 
gray  or  yellow  one ;  in  this  case  it  is  probably  not  a  true  Saccharomyces 
but  a  yeast  without  endospores.  However,  Hansen  l  has  observed  a 
rose-colored  yeast  which  did  produce  endospores.  The  dimension  of 
yeast  cells  varies  between  from  1  to  4  or  5  ^  in  width  and  from  1  to  5 
or  9  /x  in  length.  There  is  a  great  difference  in  the  cells  of  the  same 
species.  The  yeasts  are  very  polymorphic  and  are  capable  of  assuming 

1  Hansen,  E.  C.     1879.    Comp.  Rend,  des  trav.  du  lab.  de  Carlsberg,  24. 


FORMS   OF   CELLS 


T  OTTt't/Cco 

visiae 
Hansen). 


(after 


different  forms,  depending  upon  the  medium  in  which  they  are  culti- 
vated and  their  age.  It  is  thus,  for  example,  in  old  cultures  that  the 
wider  cells  diminish  generally  at  the  expense  of  the  longer  ones.  The 
different  species  of  yeasts  have  somewhat  the  same 
shape  and  are  distinguished  with  some  difficulty  from 
one  another.  If  S.  cerevisiae  is  compared  with  S. 
Pastorianus  or  S.  ellipsoideus,  quite  noticeable  differ- 
ences are  apparent.  While  S.  cerevisiae  usually  pre- 
sents round  cells  and  S.  ellipsoideus  egg-shaped  cells, 
S.  Pastorianus  presents,  to  the  contrary,  elongated 
cells,  often  in  the  shape  of  a  sausage.  But  besides 
these  elongated  forms,  one  may  find  in  cultures  round 
cells  which  may  scarcely  be  differentiated  from  S. 
cerevisiae  or  S.  ellipsoideus.  On  the  other  hand,  in 
culture  of  S.  cerevisiae  and  S.  ellipsoideiis  may  be  found  round  cells, 
and  also  elliptical  cells  which  bear  much  resemblance  to  S.  Pastorianus. 
It  is  thus  apparent  that  these  three  species  may 
not  be  closely  differentiated  by  the  shape  of  the 
cells.  There  is  always  a  predominating  form 
which  attracts  attention;  with  S.  cerevisiae  the 
predominating  form  is  round  ;  with  S.  ellipsoideus 
it  is  elliptical,  while  with  S.  Pastorianus  it  is 
most  frequently  elongated. 

The  majority  of  the  yeasts,  notably  those  of 
industrial  importance  (beer,  alcohol,  wine  and 
cider),  present  a  mixture  of  spherical  and  elon- 
gated cells.  Although  this  is  the  case,  a  predominating  form  exists 
which  may  be  of  three  types,  the  cerevisiae  type,  the  ellipsoideus 
type  or  the  Pastorianus  type. 

Among  the  yeasts,  which  are  very  numerous  and  in  which  the  cell 
shapes  are  variable  and  in- 
definite, are  often  found 
certain  species,  or  groups 
of  species,  in  which  the  cells 
present  a  characteristic 
shape  and  which  are  sepa- 
rated closely  from  the  pre- 
ceding yeasts.  Hansenia 
apiculata,  for  example,  of- 
fers cells  which  are  usually 
of  the  shape  of  a  lemon,  being  provided  with  small  projections  from 
which  the  name  apiculata  is  derived.  A  series  of  species  of  yeasts  is 
known  which  possesses  a  similar  shape  and  these,  without  doubt,  are 
varieties  of  Hansenia  apiculata  or  neighboring  species.  (See  Fig.  4,  g.) 


Fig.  3.  —  Saccharomyces 
Pastorianus  (accord- 
ing to  Hansen). 


Fig. 


4.  —  Showing    the    Different    Shapes    of 
Yeast  Cells. 


a,  cerevisiae  type;  b,  ellipsoideus;  c1,  c2,  Pastorianus  type; 
d,  Mycoderma  type;  e,  f,  Torula  type;  g,  apiculate 
type;  h,  Saccharomy  codes  type;  i,  Schizosaccharomyces; 
/ 1,  k2,  Mycelial  structures ;  I,  ml,  m2,  m3,  Amoeboid  forms 
(according  to  Lindner). 


8        MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 

Most  of  the  Torula  are  easily  recognized  by  their  almost  perfectly 
round  shape,  their  content  of  fat,  their  peculiar  manner  of  propagation 
which  causes  them  to  give  off  simultaneously  many  small  round  buds, 
and,  finally,  by  their  membrane,  almost  always  surrounded  by  a  layer 
of  a  mucilaginous  substance.  The  genus  Torulaspora  and  a  few  other 
yeasts  have  this  same  shape,  which  is  known  as  the  Torula  type. 

The  Mycoderma  and  certain  members  of  the  genus  Pichia  often 
possess  a  decidedly  refractive  appearance,  and  elongated  cylindrical 
cells  which  bud  almost  exclusively  at  two  poles;  their  contents  is  trans- 
parent, enclosing  a  number  of  refractive  granules  localized  especially 
in  the  extremities.  This  is  the  mycoderma  type  of  yeast  cell. 

With  S.  Ludwigii,  the  cells  possess  a  very  peculiar  form,  tubuliform, 
bottle,  or  sausage  shaped.  Their  division  is  intermediary  between 
budding  and  partition. 

Finally  there  are  the  Schizosaccharomyces,  which  are  not  ordi- 
narily confused  with  other  yeasts,  for  as  the  name  indicates,  they 
always  multiply  by  division  and  not  by  budding.  In  Sch. '  octosporus 
the  cells  vary  from  a  spherical  form  to  the  form  of  a  drum  stick.  The 
spherical  cells  resemble  huge  micrococci  while  the  drum-stick-shaped 
cells  resemble  the  bacilli. 

It  is  seen,  then,  that  the  yeasts  present  very  common  forms  which 
are  exhibited  rather  regularly  by  the  various  species.  It  is  well  to 
add  that  certain  yeasts  are  able  to  assume  abnormal  forms.  Thus, 
Lindner  showed  that  S.  Bailii,  when  growing  in  giant  cultures  on 
gelatin,  resembled  ameboid  bodies. 

Mycelial  Formations 

It  has  been  stated  before  that  the  yeasts  may  grow  in  a  filamen- 
tous or  mycelial  formation.  Nevertheless  it  does  not  occur  in  all 
of  the  yeasts,  and  never  appears  where  there  is  feeble  development. 
It  appears,  however,  only  under  special  conditions. 

Mycelial  formation  was  observed  for  the  first  time  by  Hansen 
in  the  growth  which  covered  the  surface  of  fermenting  liquids;  this 
is  termed  a  pellicle  or  scum.  This  growth  presents  a  very  different 
appearance  from  that  which  is  found  upon  the  bottom  of  flasks. 
Colonies  are  composed  of  long  threads  and  cells  and,  little  by  little, 
the  growth  takes  on  a  resemblance  of  a  mycelium.  The  formations, 
however,  always  remain  in  a  rudimentary  state. 

The  investigations  of  Hansen,  Lindner,  and  Will  have  shown  that 
certain  yeasts  are  equally  capable  of  forming  the  mycelial-like  struc- 
ture when  growing  on  gelatin.  It  manifests  itself  very  well  in  S. 
marxianus  and  carlsbergensis,  Pichia  membranaefaciens,  in  Zygo- 


CELL  DIVISION  9 

saccharomyces  priorianus  and  japonicus  and  S.  Ludwigii.  In  this  last 
variety,  Hansen  has  proved  the  production  of  a  well-developed  my- 
celium; however,  this  mycelium  is  rarely  composed  of  elements  which 
are  solidly  united.  The  cells  are,  however,  separated  by  well-marked 
walls,  and  each  is  able  to  thrust 
out  buds  or  to  develop  ascospores. 
Certain  parts  of  the  mycelium  offer 
very  abnormal  forms. 

With  certain  species,  the  growth 
which  is  formed  at  the  bottom  of 
a  flask  during  fermentation  has  a 
tendency  to  produce  filamentous 
formations.  Thus  with  S.  marxianus 
has  been  observed  the  formation 
of  little  flakes  of  mycelium  which 
rest  on  the  bottom  of  the  flask  or 
float  lightly  in  the  liquid.  Recently 
Lepeschkin1  secured  with  Sch. 
Pombe  and  mellacei,  under  certain 
conditions,  the  formation  of  little  Fig<  5._Mycelial  Formations  in  Old 
flocks  presenting  all  of  the  charac-  Scums  of  Saccharomyces  Ludwigii 


teristics  of  a  mycelium. 


(according  to  Hansen). 


Mycelial  formations  are  found  well  developed  in  a  yeast  described 
by  Guilliermond  in  1917  under  the  name  of  yeast  from  Pulque  No. 
2.2  A  typical  mycelium  was  formed  of  budding  yeasts  in  most  media, 
especially  in  the  sediment  which  formed  in  beer  wort  as  well  as  in  the 
flocculent  particles  which  float  in  the  medium,  on  slices  of  carrot,  and 
beer  wort  agar.  The  ascs  seem  to  appear  indifferently  from  the  yeasts 
and  from  units  in  the  mvcelium. 


CELL  DIVISION 

(A)  Budding 

Practically  all  of  the  yeasts  divide  by  budding;  it  is  the  charac- 
teristic method  for  multiplication.  The  bud  appears  as  a  little 
prominence  separated  from  the  wall  of  the  mother  cell  by  a  very 
narrow  collar.  Little  by  little  it  increases  in  size.  When  it  has 
acquired  a  certain  size,  always  smaller  than  the  mother  cell,  it  sepa- 

1  Lepeschkin,  W.     1903.     Zur  Kenntnis  der  Erheblichkeit  bei  der  einzelligen 
Organismen.     Cent.  Bakt.  Abt.  II,  10. 

2  Guilliermond,  A.    Levaduras  del  Pulque.    Boletin  de  la  Direccion  de  Estudios 
Biologicos.     Mexico,  ii,  1917. 


10      MORPHOLOGY  AND   DEVELOPMENT  OF  YEASTS 

rates.  The  daughter  cell  increases  in  size  and  soon  equals  that  of 
the  mother  cell  after  which  it,  in  turn,  buds. 

As  has  been  said  above,  when  multiplication  is  very  active,  each 
cell  forms  many  buds  simultaneously  on  different  parts  of  its  sur- 
A     A  Q        Q  face*     -^   may   naPPen  that  the   buds 

'9  o"X   fcQ,,6'N&  b<^P          attached  to  the  cell  which  gave  birth 
d<>    ^^     ^         to  them  may  begin  to  bud   before  an 
<K    ^\d    6"        absolute  breaking  apart  has  taken  place. 
^    e**  e**   "^         This  resuits  fn  the  formation  of  a  small 
,cQo   •J$XC*^%""'       colony  which  is  made  up  of  a  number  of 
y          ^^  r^  \?        adhering    cells.    Depending    upon    the 
,,       species,  the  cells  appear  united  two  by 

^  g"->P       *     A**'    I?"      two'  Producing  a  colony  made  up  of  15 
'0    >5     (3  Q        or  20  cells.     In  general,  top  yeasts  are 

*1    l\    l\    f\ ''  fi\        distinguished  from  the  bottom  yeasts 
-       p '     „        by   the   fact   that   the   former  remain 
j>   ^  ^        united   to   one  another,  forming  little 
chains,  while  the  latter  separate. 

With  s-  «^tos  and  the  senus 

Hansenia  we  have  seen  that  the  cells 

are  provided  at  one  or  both  of  the  extremities  with  little  projections 
which  give  them  the  appearance  of  a  lemon.  It  is  interesting  to 
observe  how  budding  is  accomplished  in  this  yeast.  Hansen  has 
shown  that  the  buds  always  form  at  the  extremity  sof  the  cell.1 
The  young  bud  may  be  apiculate  at  its  free  extremity,  but  it  may  be 
oval  and  give  birth  to  oval  buds  deprived  of  points.  This  may  be 
lost  and  the  property  of  forming  points  again  assumed. 

(B)  Transverse  Division 

The  genus  Saccharomyces  presents  a  form  of  transition  between 
the  ordinary  yeasts,  which  divide  by  budding,  and  the  Schizo- 
saccharomyces,  in  which  division  is  accomplished  transversely.  In  the 
Saccharomyces  division  consists  of  a  sort  of  budding  accompanied 
by  the  formation  of  a  transverse  partition,  i.e.,  a  process  intermediary 
between  budding  and  partition.  The  cells  bud  generally  at  their 
extremities;  this  is  exceptional  only  when  lateral  budding  is  proven. 
Multiplication  is  often  accomplished  in  the  following  manner:  The 
cell  elongates  and  at  one  end  a  sort  of  tube  puffs  out.  This  enlarges 
and  is  transformed  slowly  into  a  bud  which  remains  united  to  the 
cell  by  a  wide  collar.  A  wall  is  formed  across  this  which  separates 
the  cells  from  the  bud. 

1  Hansen,  E.  C.    Comp.  Rend,  des  trav.  du  lab.  de  Carlsberg,  3,  1881. 


CELL  DIVISION 


11 


Fig.  7.  —  Budding 
in  Saccharomycodes 
Ludwigii. 


True  transverse  partition  is  met  only  with  the  Schizosaccharomyces 
of  which  we  shall  mention  the  principal  species. 

Sch.  octosporus  possesses  round  or  oval  cells;  in  young  cells  the 
oval  form  predominates.  They  elongate  and,  after  having  acquired 
a  certain  size,  form  a  wall  across  the  middle.  This 
splits  apart  and  the  two  cells  become  rounded. 
They  elongate  when  they  have  achieved  their 
growth,  and  finally  separate  completely;  but  often 
the  two  cells,  though  remaining  attached,  undergo 
a  new  partition  which  makes  two  daughter  cells. 
Thus  a  row  of  cells  is  secured  which  are  arranged 
parallel  to  each  other.  Sometimes,  when  multi- 
plication is  very  rapid,  a  primary  transverse  wall  is  formed  which 
makes  two  cells;  without  separating,  these  produce 
another  partition.  In  this  manner,  small  filaments 
may  be  found  which  eventually  break  apart. 

At  the  end  of  some  culture  periods  and  also 
under  certain  conditions  the  cells  show  a  tendency 
to  take  spherical  forms.  In  this  case  partition  is 
accomplished  in  the  same  manner;  but  it  also  often 
happens  that,  on  account  of  rapid  multiplication, 
the  two  cells  set  apart  by  a  partition  remain  at- 
tached without  rounding  their  adjacent  planes. 
Each  may  then  form  transverse  partitions  which 
form  two  new  cells, 
(according  Such  an  arrange- 
ment resembles  the 
sarcina  grouping.  In  this  latter  case  par- 
tition is  accomplished  in  two  directions. 
The  daughter  cells  remain  associ- 
ated for  some  time,  giving  the  appear- 
ance of  colonies.  These  colonies 
present  different  appearances,  depend- 
ing upon  the  age  of  the  culture.  In 
the  early  stages  of  their  development 
these  colonies  are  composed  of  elongated 
cells.  Later  muriform  cells  are  apparent.  Fig.  9.  —  Partition 

In  the  other  Schizosaccharomyces 
(Sch.  Pombe  and  Sch.  mellacei),  in  which 
the  cells  look  like  drum  sticks,  division  is  accomplished  in  the  same 
manner.  When  they  have  acquired  their  maximum  size,  the  cells 
form  a  partition,  which  divides  them  into  two  cells.  They  either 
separate  immediately  or  remain  attached  for  some  time. 


Fig.  8.  —  Showing 
Partition  in  Schizo- 
saccharomyces octo- 
sporus 
to  Schionning). 


Pombe 


in    Schizosac- 
(according   to 


12      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 


In  the  latter  case,  they  are  able  to  turn  about  and,   remaining 

attached,  undergo  a  transverse  division 
as  with  Sch.  octosporus.  They  often  re- 
main completely  adherent  to  form  a 
chain  with  2,  3,  or  4  elements.  Under 
certain  conditions,  notably  in  an  en- 
vironment with  too  little  air,  the  cells 
show  a  very  marked  tendency  of  adhering 
together  in  chains  which  branch. 

Sulc 1    has    proven   the  existence   in 

Fig~lO.-  Doable  Cells.    The    certain    ^Uzosaccharomyces    of     certain 
Membraneous  Layer  is  Partly    fatty  bodies.      These  Schizosaccharomyces 
(aC"   muMPly    indifferently   by   partition   or 
budding.    This  should  be  confirmed. 


Durable  Cells 

In  the  pellicle  which  appears  after  a  period  of  time  on  the  surface 
of  nutrient  media,  and  in  deposits  at  the  bottom  of  flasks  containing 
certain  special  media  (sugar 
solutions  containing  tartaric 
acid  or  citric  acid  and  mineral 
matter),  Will2  has  observed 
cells  which  possess  thick  walls 
and  whose  contents  are  rich 
in  glycogen  and  fats. 

From  the  researches  of 
Will  and  Casagrandi,3  these 
cells  possess  a  double  mem- 
brane; the  outer  one  is  very 
fragile  and  easily  broken  to 
pieces.  These  membranes  are 
made  more  visible  by  treating 
the  cell  with  osmic  acid  or  by 
the  Ripart-Petit  fluid  (hydro- 
chloric and  chromic  acids,  1 
per  cent). 

Will  has  called  these  cells 


11.  —  Germination    of    Durable    Cells 
(according  to  Will). 


1  Sulc,    K.      "Pseudovitellius"   und  ahnliche   Gewebe   der  Homopteren   sind 
Wohnstatten  symbiotischer  Saccharomyceten  (Sitzungsberichte  der  Konigl.  Bohm. 
Gesellschaft  der  Wissenschaften  in  Prag.     March  30,  1910). 

2  Will,   H.     Vergleichende  Untersuchungen  an  vier  untergarigen  Arten  von 
Bierhefe.    Zeitschr.  f.  d.  ges.  Brauwesen,  Munich,  II,  18,  1895. 

3  Casagrandi.    1897.    Ueber  Morphologic  der  Blastomyceten.    Cent.  Bakt.,  3. 


SPORULATION  13 

durable  cells  (Dauernzellen) ;  he  regards  them  as  resistant  organs 
which  serve  to  perpetuate  the  species  over  unfavorable  periods.  In 
this,  they  are  similar  to  the  ascospores.  Perhaps  they  may  be 
regarded  in  the  same  light  as  cysts,  or  clamydospores,  which  have 
been  observed  so  frequently  in  the  Endomyces. 

When  these  durable  cells  are  placed  again  in  favorable  circum- 
stances, their  membrane  is  broken  and  budding  takes  place  giving 
rise  to  spherical  and  elongated  yeasts,  be  they  separated  or  in  groups. 

Sporulation 

Sporulation  is  a  form  of  resistance  which  allows  the  yeast  to 
remain  viable,  even  though  active  budding  has  stopped.  It  plays 
an  important  r61e  in  the  hibernation  of  yeasts,  permitting  them  to 
pass  the  winter  in  the  ground  of  vineyards  where  they  are  deposited 
in  the  autumn.  Sporulation  is  observed  in  old  cultures  where  food 
is  scarce,  also  in  certain  solid  media  such  as  carrots  or  gelatin  which 
are  not  very  favorable  for  budding.  It  is  especially  easy  to  secure 
Sporulation  by.  submitting  the  yeasts  to  starvation  after  they  have 
been  able  to  build  up  sufficient  reserve  products  necessary  for  the 
formation  of  ascospores. 

We  shall  take  up  in  a  following  chapter  the  details  which  determine 
Sporulation;  therefore  this  question  will  not  receive  attention  at 
this  time. 

Internal,  or  ascospores,  were  observed  for  the  first  time  by  Schwann 
in  1839  and  described  by  Seynes.  They  were  regarded  by  some 
authors,  notabty  Van  Tieghm,  as  resulting  from  a  sort  of  encystment 
resulting  from  some  pathological  process.  Brefeld  considered  these 
cells,  which  bear  spores,  as  sporangia  or  cysts.  On  the  contrary, 
Rees,1  de  Bary,2  and  later  Hansen,3  likened  the  sporangia  of  yeasts 
to  the  asc  of  the  Ascomycetes  and  regarded  the  yeasts  as  a  group  of 
fungi.  This  opinion  has  been  entirely  confirmed  by  our  investiga- 
tions on  the  cytological  phenomena  of  the  formation  of  ascospores, 
and  especially  by  the  discovery  in  certain  yeasts  of  a  copulation 
in  the  origin  of  the  asc.  It  is  definitely  admitted  today. 

Sporulation  is  indicated  by  any  cell,  either  yeast  cell  or  a  cell 
constituting  a  rudimentary  mycelium.  In  this  way  certain  yeasts 
(S.  Ludwigii,  Pichia  membranaefadens)  are  able  to  form  ascospores 
in  mycelial  cells  developing  on  the  surface  of  old  cultures.  Each 
cell,  then,  seems  able  to  develop  into  an  asc. 

1  Rees,  H.  1870.  Botan.  Untersuchungen  iiber  die  Alcoholgarungspilze,  Leipzig. 

2  De  Bary,  A.    Morphologic  des  Pilzes.    Leipzig,  1866. 

3  Hansen,  E.  C.    Recherches  sur  la  physiologic  et  la  morphologic  des  ferments 
alcooliques.    Comp.  Rend,  des  trav.  du  lab.  de  Carlsberg,  2,  1883. 


14      MORPHOLOGY  AND  DEVELOPMENT  OF   YEASTS 

With  the  exception  of  the  case,  which  we  shall  consider  a  little 
later,  in  which  the  asc  results  from  a  copulation,  the  ascs  retain 
generally  the  form  and  dimensions  of  ordinary 
cells.  (Fig.  12.)  However,  in  Nematospora 
coryli  and  Monospora  cuspidata  the  ascs  are 
rectangular  cells  more  elongated  and  larger  than 
Fig.  12. — Ascs  of  Sac-  the  vegetative  cells. 

charomyces     cerevisiae          often  tne  ascs  are  derived  from  cells  which 
(according  to  Hansen). 

have  not  ceased  to  bud.  There  are  no  clear- 
cut  limits  between  budding  and  sporulation,  for  both  are  able  to  be 
carried  on  at  the  same  time.  Budding  continues  and  slows  up  only 
at  the  time  when  sporulation  begins.  This  explains  why  one  often 
sees  cells  with  ascospores  and  buds,  the  bud  remaining  attached  to 
the  mother  cell  and  developing. 

The  number  of  ascospores  contained  in  an  asc  is  variable.  It 
may  vary  between  1  and  12.  However,  it  usually  becomes  fixed 
as  in  many  of  the  industrial  yeasts  where  a  certain  number  usually 
predominates. 

The  number  of  ascospores  in  S.  cerevisiae  varies  between  1  and 
5,  but  4  are  more  frequent.  With  S.  Pastorianus  the  same  variation 
obtains,  but  2  ascospores  are  more  common.  With  other  yeasts,  the 
number  varies  less  widely  and  is  more  constant.  With  Saccharomyces 
Ludwigii  and  the  yeast  Johanisberg  II,  it  is  almost  always  4.  With 
Sch.  octosporus,  sometimes  4  and  sometimes  8  ascospores  may  be 
counted.  With  Sch.  mellacei  and  Pombe  the  number  of  ascospores 
is  invariably  4.  The  same  number  obtains  constantly  with  Nema- 
tospora coryli.  Monospora  cuspidata  contains  only  a  single  spore  in 
the  asc.  With  Debaryomyces  globosus  and  Schwanniomyces  occiden- 
talis  the  number  varies  between  1  and  2.  Thus,  with  each  yeast  the 
asc  tends  to  form  a  constant  number  of  ascospores.  This  number 
varies,  depending  upon  the  species,  from  1  to  2,  4,  and  rarely  8  or 
more. 

The  ascospores  have  dimensions  between  1.5  and  5  ju.  Usually 
they  are  spherical  or  oval  (S.  cerevisiae,  S.  Pastorianus,  S.  ellip- 
soideus).  Sometimes  they  possess  a  globule  of  fat.  The  ascospores 
of  some  yeasts  have  characteristic  forms.  In  Willia  anomala,  also  in 
the  genus  Hansenia,  the  ascospores  present  a  form  quite  similar  to 
the  cells  of  lower  Ascomycetes  (Ascoidea  rubescens,  Endomyces  deci- 
piens,  and  Endomyces  fibuliger) ;  they  are  hemispherical  and  their 
adjacent  planes  are  provided  with  a  projecting  border  which  gives 
them  the  appearance  of  a  hat.  The  ascospores  of  Willia  Saturnus 
have  the  shape  of  a  lemon  and  are  girdled  with  a  projecting  ring. 
(Fig.  13,  3.)  Cells  of  Pichia  membranaefaciens  have  irregular  shapes. 


SEXUALITY  15 

spherical,  oval,  elongated,  triangular,  kidney-shaped,  or  hemispherical. 
Sometimes  they  are  small  and  hyaline,  with  a  refractive  globule  in  the 
center.  In  Debaryomyces  and  Nadsonia  one  finds  globular  ascospores 
enveloped  in  a  membrane  which  is  covered  with  stiff,  erect  pro- 
tuberances. (Fig.  13,  5.)  In  Schwanniomyces  occidentalis,  the  asco- 
spores are  provided  with  a  projection  about  the  cells  which  divides 
them  into  two  unequal  parts.  (Fig.  13,  6.)  The  ascospores  of  Schizo- 
saccharomyces  may  be  ellipsoidal  or  elongated,  and  their  membrane 
is  impregnated  with  starchy  materials  which  are  stained  blue  with 
iodin-potassium  iodide  solution.  Nematospora  coryli  possesses  asco- 
spores which  are  long  and  fusiform;  they  are  provided  with  long 


O  7(3  0  QOGpi 

'  V  f 


1234  "'5 


Fig.  13.  —  Showing  the  Various  Shapes  of  Ascospores  in  Yeasts. 

1,  Ascospores  from  S.  cerevisiae;  2,  Ascospores  from  Willia  anomala;  3,  Asco- 
spores from  Z ygosaccharomyces  chevalieri;  4,  Ascpspores  from  Willia  Sa- 
turnus;  5,  Ascospores  from  Pichia  membranaefaciens;  6,  Ascospores  from 
Debaromyces;  7,  Ascospores  from  Schwanniomyces  occidentalis;  8,  Ascospores 
from  Saccharomyces  capsularis;  9,  Ascospores  from  Nematospora  coryli;  10, 
Ascospores  from  Monospora  cuspidata;  11,  Ascospores  from  Coccidiascus  Legeri. 

structures  at  their  extremities  which  are  similar  to  cilia.  Monospora 
cuspidata  also  has  long  cells  which  look  much  like  needles.  (Fig. 
13,  9.) 

Most  of  the  yeasts  known  possess  only  a  single  membrane.  In 
S.  guttulatus,  there  are,  on  the  contrary,  two  membranes.  The 
outer  one  breaks  at  the  moment  of  germination. 

SEXUALITY 
(A)    Copulation  Preceding  the  Formation  of  the  Asc 

Recent  investigations  have  shown  that  with  a  certain  number  of 
yeasts,  the  asc  results  from  a  copulation  which  closely  resembles  that 
of  cells  which  one  finds  in  certain  of  the  lower  Ascomyces.  (Eremascus 
and  Endomyces.) 

It  was  among  the  Schizosaccharomycetes  that  this  phenomenon 
was  noticed  for  the  first  time.  In  this  group  of  yeasts,  characterized 
as  we  have  seen  by  a  special  multiplication  of  cells  which  occurs 
always  by  transverse  partition,  only  a  few  species  are  known  (Sch. 
octosporus,  Sch.  Pombe,  and  Sch.  mellacei).  These  three  species  show 
sexual  processes. 


16      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 


In  1895,  Schionning1  showed  in  a  short  note  that  the  ascs  of 
Sch.  octosporus  resulted  from  the  fusion  of  two  sister  cells,  but  not 
having  observed  the  cytological  phenomena  which  accompanied 
this  fusion,  he  was  not  able  to  realize  its  significance.  Hoffmeister2 
thought  that  he  observed  in  this  phenomenon  a  nuclear  fusion,  but 
at  that  time  the  nutrition  of  yeasts  was  insufficiently  known  to 
permit  accurate  observations.  Bearing  in  mind  the  investigations 
of  these  two  investigators,  Guilliermond 3  has  succeeded  in  demon- 
strating that  this  fusion  is 
a  true  copulation.  It  is 
easy  to  observe  this  phe- 
nomenon in  a  Bottcher 
moist  chamber  in  a  drop  of 
beer  wort  gelatin.  The 
ascospores  deposited  in  it 
are  not  slow  to  germinate 
and  produce  vegetative 
cells  which  multiply  very 
actively  during  the  first 
two  days;  toward  the  third 
Fig.  14.  —  Different  Stages  in  the  Development  day  the  multiplication  de- 
of  Schizosaccharomyces  octosporus.  creases.  The  cells  are 

a,    and  h,  germination   of  ascospores;     c,   multiplication   of         .  ,,  .          V.LJ.I 

cells;  d,  copulation  and  formation  of  the  asc;   e,  f,  g,  and  k,       tnen      adherent      in       little 
copulation  in  a  species  tending  to  become  asporogenic.  ,  ..  _ 

colonies  of  15  or  20,  per- 
haps a  few  less.  Some  continue  to  divide,  but  most  cease  to  multiply. 
At  this  moment  copulation  commences  and  is  accomplished  in 
the  following  manner:  Two  cells  identical  in  characteristics  and 
lying  adjacent  to  each  other  in  the  same  colony  are  joined  by  means 
of  a  copulation  canal,  formed  by  the  fusion  of  two  little  projections 
put  out  by  each  cell.  (Fig.  15.)  The  middle  wall  which  separates 
the  two  cells  is  rather  quickly  dissolved,  and  the  nucleus  of  each 
cell,  transformed  thus  into  gametes,  passes  through  the  copulation 
canal.  By  this  operation,  a  single  cell,  which  is  an  egg  or  zygospore, 
is  formed.  Formed  in  this  manner  by  isogamic  copulation,  the  egg 
soon  germinates.  It  increases  in  volume,  while  its  nucleus  under- 
goes two  successive  divisions,  sometimes  three,  which  give  4  or 
8  nuclei.  Then  these  become  distributed  about  the  zygospore  and, 

1  Schionning,  H.   1895.   Nouvelle  et    singuliere    formation    d'ascus  dans   line 
levure.    C.  R.  lab.  de  Carlsberg,  4. 

2  Hoffmeister,  C.     Zum  Nachweiss  des  Zellkerns  bei  Saccharomyces.    Sitzungs- 
ber.  deutsch.  naturw.  mediz.  Vereins  f.  Bohmen,  25,  1900. 

3  Guilliermond,  A.     Recherches  histologiques  sur  la  sporulation  des  Schizo- 
saccharomycetes.     Comp.  Rend.  Acad.  Sci.  133,  July  22,  1901.     Recherches  cyto- 
logiques  sur  les  levures,  Storck,  ed.  Lyon,  1902. 


SEXUALITY 


17 


surrounding  themselves  with  a  zone  of  protoplasm,  form  4  or  8 
ascospores.  The  zygospore  is  then  transformed  into  an  asc.  In 
many  cases  the  fusion  of  gametes  is  not  always  complete  and  the 
asc  retains  a  median  constriction,  a  remnant  of  the  copulation  canal. 
It  often  happens  that,  in  certain  cases,  the  gametes  remain  individ- 
ualized, and  the  asc  may  be  constituted  of  two  cells  united  by  a  copu- 
lation canal.  In  this  case,  the  ascospores  are  formed  in  groups  of 
2  or  4  in  each  cell.  These  are  formed 
especially  when  copulation  takes  place 
between  cells  which,  not  being  united, 
are  obliged  to  send  out  long  projections. 
With  Sch.  octosporus  all  of  the  steps 
between  complete  and  incomplete  fusion 
of  gametes  may  be  observed,  but  in  the 
two  cases  the  result  is  the  same  and  it 
produces  a  zygospore. 

In  a  few  rare  cases  the  asc  originates  by  parthenogenesis;  this  is 
happening  when  one  sees  two  gametes  united  by  a  canal  in  which  the 
wall  has  not  been  perforated,  forming  individually  a  parthogenetic  asc. 

A  very  peculiar  fact,  and  one  which  should  be  mentioned  here 
on  account  of  its  biological  significance,  is  the  copulation  between 
two  adjacent  parent  cells  or,  as  Schionning  has  described,  between 


Fig.  15.  —  Various  Stages  in  the 
Copulation  of  Schizosaccharo- 
myces  octosporus  as  Observed 
in  Bottcher's  Moist  Chamber. 

a,  10  o'clock  in  the  morning;  b,  1 
o'clock;  c,  2  o'clock;  d,  5  o'clock; 
e,  6  o'clock. 


Fig.    16.  —  Copulation   and   Formation   of   the   Asc   in 
Schizosaccharomyces  octosporus  (in  Stained  Preparation). 

two  cells  sprung  from  the  same  mother  cell  —  between  two  brother 
gametes.  It  is  a  primitive  characteristic  which  distinguishes  this 
copulation  from  the  sexuality  of  more  highly  developed  organisms. 

As  a  rule,  almost  all  of  the  cells  fuse  two  by  two  with  the  forma- 
tion of  the  egg  and  more  often  between  two  adjacent  cells  in  the 
same  colony.  As  each  colony  is  composed  of  15  or  20  cells,  the 
gametes  are  necessarily  closely  related.  The  same  thing  is  true  in 
colonies  made  up  of  2  or  3  cells  which  are  able  to  copulate  two  by 
two.  One  is  able  to  follow  under  the  microscope  the  formation  of 


18      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 


a  daughter  cell  which  separates  from  the  mother  cell.  This  may 
unite  with  another  daughter  cell  from  the  same  parent  to  form  an 
asc.  However,  as  we  shall  see  further  on,  the  ascospores  of  the  same 
asc,  when  in  an  environment  unfavorable  to  multiplication,  fuse  two 
by  two  and  are  transformed  into  ascs  without  preliminary  biparti- 
tion.  In  this  case,  as  the  asc  always  contains  4  or  8  ascospores,  the 

gametes  will  not  be  separated  by 
more  than  3  or  4  generations. 

Copulation  may  also  be  brought 
about  between  cells  of  very  different 
parentage.  As  we  shall  see  further 
on,  Beijerinck  has  shown  that  Sch. 
octosporus,  in  continued  cultivation 
in  artificial  media,  may  lose  its 
properties  of  forming  ascospores 
in  the  and  becomes,  after  a  long  time,  an 
asporogenous  organism.  In  cultures 
undergoing  this  change,  the  num- 
ber of  asporogenous  cells  becomes  greater  and  greater  at  the  expense  of 
the  sporogenous  cells.  It  happens  that  these  latter  cells 
are  isolated  in  colonies  in  which  all  of  the  other  cells 
have  lost  the  sporogenic  function.  These,  then,  have 
to  go  to  other  colonies  in  their  vicinity  for  sporogenous 
cells  with  which  they  are  able  to  anastomose.  They 
send  out  long  tubes.  These  often  go  astray,  form  a 
wall  across  themselves  and  dissociate.  From  these 
facts,  it  is  apparent  that  the  parentage  of  the  gametes 
is  of  little  importance;  as  a  rule  gametes  more  or  less 
closely  situated  fuse  and  copulation  follows  the  law  of  Fig.  18.  —  Dif- 


Fig.    17.  —  Different   Stages 
Copulation   and   Formation  of  the 
Asc  in  Schizosaccharomyces  Pombe. 


ferent  Stages 
of  Copulation 
in  Schizosac- 
charomyces 
Pombe  as  Ob- 
served in  Bot- 
tcher's  Moist 
Chamber. 


least  resistance. 

In  Sch.  Pombe  and  Sch.  mellacei,  two  related  species, 
copulation  takes  place  in  the  same  manner  as  in  .Sch. 
octosporus,  with  the  only  difference  that  fusion  remains 
almost  always  incomplete.  The  cells  destined  to  copu- 
late are  generally  united  in  colonies  of  4  to  20  cells 
situated  in  chains  and  presenting  the  form  of  small  rods.  Copula- 
tion is  accomplished  ordinarily  between  two  cells  adjacent  in  the 
same  colony.  (Figs.  17  and  18.)  The  gametes  are  united  through  a 
canal  through  which  nuclear  and  protoplasmic  fusion  takes  place. 
(Fig.  19.)  The  nucleus  resulting  from  this  fusion  rather  quickly 
divides,  and  the  two  nuclei  thus  formed  emigrate  to  both  enlarge- 
ments of  the  zygospore  where  they  undergo  a  second  division  neces- 
sary to  the  formation  of  ascospores.  (Fig.  19.)  The  zygospore  then  is 


SEXUALITY 


19 


and  Formation  of  the  Asc  in  Schizosaccharo- 
myces  Pombe  (in  Stained  Preparation). 


transformed  into  an  asc  which  preserves  always  the  form  of  a  dumb- 
bell. The  ascospores,  to  the  number  of  4,  originate  in  pairs  in  both 
enlargements. 

Parthenogenesis,  extremely  rare  in  Sch.  octosporus,  is,  on  the 
contrary,  rather  frequent  in 
Sch.  Pombe  and  Sch.  mellacei. 
Sometimes  two  cells,  already 
united  by  a  copulation  canal, 
form,  without  reabsorbing  the 
separating  walls,  a  partheno- 

genetic  asc;  more  often  it  is  Fig.  19.  —  Various^  Stages  in  the  Copulation 
an  ordinary  cell,  which,  with- 
out trying  to  unite  itself  to 
another,  transforms  itself  directly  into  an  asc.  (Fig.  17,  4.)  Sulc  has 
described  a  new  species  of  Schizosaccharomyces,  Sch.  Aphalarae  calthae, 
found  in  the  fatty  tissue  of  the  homoptera,  which 
seems  to  present  a  copulation  analogous  to  that  of 
Sch.  octosporus.  Nakazawa  has  found  a  copulation 
quite  similar  to  that  of  Sch.  Pombe  and  mellacei  in 
Sch.  Sautanensis  and  formosensis  which  were  iso- 
lated by  him  from  sugar  products  in  Formosa. 

The  sexual  phenomena  are  present  not  only  in 
the  Schizosaccharomyces;  they  have  been  described 
also  in  a  certain  number  of  yeasts,  multiplying 
by  budding.  One  observes  not  only  isogamy  but 
heterogamy  and  intermediate  forms  between  these 
two  methods. 

Barker,1  in  1901,  established  the  first  of  these  in  a  new  species 
isolated  from  a  solution  containing  ginger,  for  which  he 
created  the  genus  Zygosaccharomyces.  This  yeast  is  known 
today  as  Zyg.  Barken.  The  copulation  is  isogamic  and 
occurs  in  the  same  manner  as  in  Sch.  Pombe  and  mel-  Fig.  21. 
lacei.  The  fusion  is  incomplete,  and  the  asc  which  results 
retains  the  form  of  two  retorts  united  by  a  collar.  The 
mixture  of  the  protoplasm  and  the  nuclear  fusion  takes 
place  in  the  copulation  canal.  The  ascospores,  which 
vary  in  number  from  two  to  four,  develop  in  both  enlarge- 
ments of  the  asc  or  exceptionally  in  but  one.  (Fig.  21.) 

Recent  work  has  shown  that  these  sexual  phenomena,  which  have 
been  regarded  as  rare  at  the  time  when  they  were  first  observed,  are 

1  Barker,  P.  A  conjugating  yeast.  Proc.  of  the  Roy.  Soc.  68,  July  8,  1901. 
On  the  spore  formation  among  the  saccharomycetes.  Jour.  Federated  Insti- 
tutes of  Brewing,  13,  1902. 


Fig.  20.  —  Copula- 
tion and  Formation 
of  the  Asc  in  Sch. 
mellacei. 


Q 


Dif- 
ferent Stages 
in  the  Copu- 
lation and 
Formation  of 
the  Asc  in 
Zygosaccharo- 
myces Barkeri 
(afterBarker). 


20      MORPHOLOGY  AND   DEVELOPMENT  OF  YEASTS 


really  quite  widespread  among  the  yeasts.  Since  the  discovery  of 
Zyg.  Barken,  Klocker  1  has  recorded  the  existence  of  a  similar  copula- 
tion in  Zyg.  Priorianus  and  Zyg.  mandshuricus,  de  Kruyff2  in 
Zyg.  javanicus,  Saito  3  in  Zyg.  japonicus,  Dombrowski 4  in  Zyg.  lactis, 
Takahashi  in  Zyg.  Major,  Richter  in  Zyg.  mellis  acidi,  and  Pearse 
and  Barker  5  in  a  yeast  from  cider  provisionally  designated  as  "  Yeast 
F."  Chatton  has  also  described  an  isogamic  copulation  in  a  yeast 
isolated  from  Drosophilia  funebris  which  he  names  Coccidiascus 
Legeri.6  It  seems  that  this  copulation  existed  among  other  yeasts  but 

had  not  received  the  atten- 
tion of  authors  who  described 
them. 

Among  all  of  these  yeasts, 
copulation  occurs  exactly  as 
in  Sch.  Porribe  and  mellacei. 
It  obtains  between  adjacent 
cells  and  very  closely  related 
parents.  With  Zyg.  Priori- 
anus,  for  example,  copulation 
almost  always  takes  place  be- 
tween two '  cells  of  the  same 
colony  made  up  of  15  or  20 
cells.  Sometimes  it  takes  place  between  cells  in  a  colony  composed  of 
only  3  or  4  cells.  It  happens  also  that  one  is  able  to  see  a  cell  in  the 
act  of  forming  a  bud  and  also  fusing  with  the  latter  before  it  has 
achieved  its  full  development.  In  this  case,  the  asc  which  results  is 
composed  of  two  unequal  enlargements;  one,  the  larger,  representing 
the  mother  cell,  and  the  other  representing  the  bud.  The  ascospores 
not  having  sufficient  space  for  germination  into  the  bud,  form  them- 
selves uniquely  in  the  mother  cell.  In  this  manner,  copulation  nor- 
mally isogamic  finds  itself  heterogamic.  Perhaps  we  may  see  in  this 
anomaly  a  tendency  to  heterogamy. 

1  Klocker,  A.,  in  Lafar  Handbuch  der  Technischen  Mykologie.      G.  Fischer, 
Jena,  1905. 

2  de   Kruyff,    E.      Untersuchungen   iiber   auf   Java  einheimische  Hefenarten, 
Cent.  Bakt.  21,  1908. 

3  Saito,  K.     Preliminary  notes  on  the  spore  formation  on  the  so-called  "Soya 
Kahmhefe."     Botanical  Mag.  13,  1909. 

4  Dombrowski,  W.     Die  Hefen  in  Milch  und  Milchprodukten.     Cent.  Bakt. 
Abt.  II,  28,  1910. 

6  Pearse,  B.,  and  Barker,  P.  The  yeast  flora  of  bottled  ciders.  Jour.  Ag. 
Sci.  3,  1908. 

6  Guilliermond,  A.  Quelques  remarques  sur  la  copulation  des  levures.  Annales 
mycologici,  8,  1910. 


Fig.  22.  —  Copulation  and  Formation  of  the 
Asc  in  Zygosaccharomyces  Priorianus. 


SEXUALITY 


21 


Another  yeast  isolated  by  Pearse  and  Barker1  from  cider  and 
designated  Yeast  G  presents  a  copulation  which  is  intermediary  be- 
tween isogamy  and  heterogamy.  In  this  species  the  two  gametes 
are  cells  of  the  same  dimensions  which  do  not  show  morphologically 
any  sexual  differentiation.  But  the  contents  of  one,  which  may  be 
regarded  as  the  male,  pass  into  the  other,  which  may  be  regarded  as 
the  female.  The  ascospores  originate  from 
this  last  and  they  are  always  to  the  number 
of  two.  (Fig.  23.) 

More  recently,  a  strictly  heterogamic  proc- 
ess was  observed  in  a  new  species  isolated  from 
fermentation  products  of  wine  from  Bili.2  This 
yeast,  which  we  have  named  Zygosaccharo- 
myces  chevalieri,  has  ascs  which  result  from  a 
copulation  between  two  cells  of  different  di- 
mensions. (Fig.  24.)  One  is  very  small  and 
represents  the  male  gamete.  It  is  young, 
while  the  other,  representing  the  female  gamete,  is  larger  and  much 
older,  having  attained  its  full  development.  The  two  cells  unite  by 

means  of  a  copulation  canal 
and  the  contents  of  the  male 
gamete  pass  into  the  female 
gamete  in  which  the  protoplas- 
mic and  nuclear  fusion  takes 
place.  After  this  has  taken 
place,  the  female  gamete  sepa- 
rates itself  by  means  of  a  wall, 
and  produces  from  1  to  4 
ascospores.  During  this  the 

Fig  24. -Various  Stages  in  the  Copulation   membrane  of  the  male  gamete 
and  Formation  of  the  Asc  in  Yeast  Bill  2. 


23.  —  Copulation    in 
Yeast    G"    of     Pearse 
and    Barker     (according 
to  Pearse  and  Barker). 


22 


23 


1-3,  gametes  sending  out  tubules  destined  for  copula- 
tion;  4-8,  union  of  two  gametes  by  a  copulation 
canal;  9-18,  the  contents  of  the  male  gamete  mixes 


*****  iis  ™mbTa™  and 


.  . 

1S  absorbed.       It   IS    alSO  rare   to 

nK™™^      Qn       oHnlt      a«n       acrmn 
ODServe      an      aClUlL      d,SC       again 

united  to  a  male  gamete. 

Recent  investigations  have 

shown    that    the    heterogamic 

copulation  occurs  frequently.  In  Zygosaccharomyces  priorianus  it  has 
been  shown  that  copulation  is  accomplished  sometimes,  but  rarely,  be- 
tween an  adult  cell  and  a  bud  formed  by  it.  In  Debar  omyces  globosus 
Guilliermond  has  shown  that  there  takes  place  a  heterogamic  copula- 

1  Pearse,  B.,  and  Barker,  P.    The  yeast  flora  of  bottled  ciders.    Jour.  Agricul- 
tural Science,  3,  1908. 

2  Guilliermond,    A.      Sur   un   exemple   de   copulation   heterogamique   observe 
dans  une  levure.    Comp.  Rend,  de  la  Soc.  Biologic,  70,  1911. 


22      MORPHOLOGY  AND   DEVELOPMENT   OF  YEASTS 

tion  between  a  mother  cell  and  a  bud  formed  by  that  cell,  which  is 
very  small  and  still  attached  to  the  mother  cell.  About  25  per  cent, 
only,  of  the  ascs  result  from  an  isogamic  copulation  between  two 
cells  of  the  same  dimensions.  In  Debaromyces  tyrocolla,  the  copula- 
tion is  accomplished  most  often  between  the  mother  cell  and  its 
bud  and  the  isogamic  copulation  is  very  rare.  According  to  Konoko- 
tin,  the  genus  Debaromyces  may  be  represented  by  a  heterogamic 
copulation.  In  one  other  yeast,  isolated  by  Guilliermond  from  oranges, 
the  Zygosaccharomyces  Nadsonii,  a  heterogamic  copulation  has  been 
described,  but  with  this  yeast  the  heterogamy  is  the  rule,  and  there 
seems  to  be  no  instances  of  isogamy. 

Finally  Nadson  and  Konokotin  have  discovered  in  the  mucous 
secretions  of  trees  two  species  of  yeasts  (Nadsonia  fulvescens  and 
elongata)  in  which  the  copulation  always  takes  place  by  heterogamy 
between  an  adult  cell  and  one  of  the  buds  formed  by  it.  All  of  the 
contents  of  the  male  gamete  go  into  the  female  gamete,  but  the 
female  gamete  does  not  transform  directly  into  an  asc.  It  gives 
birth,  by  budding,  to  a  new  cell  into  which  its  contents  are  poured, 
and  it  is  this  cell  which  becomes  the  asc,  usually  including  a 
single  ascospore.  The  authors  think  that  the  asc,  formed  by  budding, 
represents  a  rudiment  of  a  sporophyte,  and  consequently  have  es- 
tablished for  these  two  species  the  genus  Nadsonia  (Guilliermondia) . 
With  Zygosaccharomyces  it  has  been  difficult  to  determine  the  parents 
of  the  gametes  which  unite;  the  copulation  is  almost  always  ac- 
complished between  an  adult  cell  and  a  young  cell.  In  other  yeasts 
with  heterogamic  copulation,  it  takes  place  between  a  mother  cell 
and  one  of  the  cells  formed  by  budding  from  it.  In  this  case  copula- 
tion may  be  autogamic. 

Guilliermond l  has  isolated  from  the  mucous  secretions  of  chestnut 
trees  a  new  yeast,  Zygosaccharomyces  Pastori,2  which  presents  a 
heterogamic  copulation  effected  between  cells  of  unequal  sizes.  The 
female  cell  is  the  adult  while  the  other,  the  male,  has  not  attained 
its  full  development.  Sometimes  there  is  little  difference  between 
the  two  cells.  The  contents  of  one  always  pour  into  the  other 
which  is  transformed  into  the  asc.  The  asc  may  contain  from  1  to 
4  ascospores  which  are  hat-shaped  like  those  of  Willia  anomala.  This 
yeast  produces  only  a  few  ascs;  the  cells,  however,  attempt  to  unite 

1  Guilliermond,  A.      Sur   une   nouvelle   levure   a  copulation  he'te'rogamique. 
Comp.  Rend.  Soc.  Biol.  1919. 

2  This  yeast  has  not  been  fully  described  but  presents  a  resemblance  to  Willia 
anomala  in  having  hat-shaped  ascospores.     Its  cultural  characteristics,  however,  do 
not  class  it  in  the  group  Willia.      Klocker  has  described  an  apiculate  yeast  in  the 
Saccharomyces  apicidatus  group  in  which  the  spores  are  hat-shaped.     This  form 
ought  not  to  be  regarded  as  a  characteristic  of  the  group  Willia 


SEXUALITY  23 

with  others  by  means  of  small  projecting  tubes,  some  cells  possessing 
many  of  them,  but  this  is  hardly  ever  successful.  It  is  then  possible 
that  we  are  concerned  with  yeast  which  is  in  the  process  of  losing  its 
sexuality  and  sporogenic  function. 

Cesari1  has  recently  isolated  a  series  of  yeasts  from  sausage  and 
salted  meats  which  seems  to  act  on  albuminous  matter  and  play  a 
role  in  the  ripening  process.  All  of  these  yeasts  possess  a  heterogamic 
copulation  resulting  in  the  formation  of  an  asc  with  a  single  ascospore. 

(B)   Copulation  of  Ascospores  or  Parthenogamy 

The  copulation  which  has  just  been  described,  is  not  the  only 
form  of  sexuality  observed  among  the  yeasts.     In    certain    species 
occurs  a  sexually  different  process  which  is  brought  about  by  a  sub- 
sequent stage  in  the  germination  of  ascospores.     It 
is  thus  that   in   S.    Ludwigii   and    Willia   Saturnus 
and  the  yeast  Johannisberg  II,  an  isogamic  copula- 
tion    between    ascospores    originating   from    an    asc 
formed  without  fusion,  has  been  established.     This 
phenomenon  will  be  discussed  at  this  time  without 
entering  into  a  detailed  discussion  of  the  germination 
of  ascospores. 

In  S.  Ludwigii  the  asc  contains  almost  always       /~aO*~ 
four  ascospores;    at  the  moment  of  copulation,  these          vxSP3 
ascospores  copulate  two  by  two  by  means  of  a  copu- 
lation  canal  formed  by  the  fusion  of  two  little  pro- 
jections  from  each  cell.     (Fig.  25.)     It  was  Hansen2 

who  showed  for  the  first  time  the  existence  of  this  Fig.      25.  —  Vari- 

!  T ,     •        i  i    ,         ,  i          ,i  •      e  ous  Stages  in  the 

phenomenon.     It    is    shown    later   that   this  fusion      Germination    of 

presents  the  characteristics  of  true  copulation  and  Ascospores  in  S. 
that  it  is  accompanied  by  a  nuclear  fusion.  servedm ^Moist 

The  nucleus  and  cytoplasm  are  introduced  into       Chamber       (ac- 
the   copulation   canal  and  it  is  there  that  the  mix-      sen)"18 
ture  of  nucleus  and   cytoplasm  takes  place.     The 
fusion  remains  incomplete  and  the  zygospore  is  formed  from  two  asco- 
spores united  by  a  copulation  canal.     In  this  canal,  the  egg  is  formed 
which  brings  about  the  germination  of  the  zygospore.     It   elongates 
into  a  germination  tube  from  which  originate  numerous  vegetative  cells. 

Copulation  is  accomplished  normally  between  two  ascospores  of 
the  same  asc  before  the  partition  is  absorbed.  Buds  result  from  the 

1  Cesari,  E.-P.     La  maturation  du  saucisson.     Comp.  Rend.  Soc.  Biol.,  1919. 

2  Hansen,  E.  C.    Recherches  sur  la  morphologic  et  la  physiologic  des  ferments 
alcooliques,  3,  1891. 


24      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 


germination  of  the  zygospore  which,  in  developing,  perforate  the  wall 
of  the  asc.  The  copulation  of  the  asc  is  always  by  autogamy  and, 
as  each  asc  contains  only  four  ascospores,  it  will  be  able  to  occur 
only  between  sister  ascospores.  However,  by  force  of  circumstances 
copulation  may  also  be  accomplished  between  ascospores  from  dif- 
ferent ascs  and  consequently  from  more  distant  relationships.  This 

is    almost     constantly 

>\  _ -ZZ?S?^^      xJr^C«TV^)        /^^  ~\.*/°^->-^  ~~~4.         :±l* 


met  with  when  one 
makes  old  ascospores 
germinate;  under  these 
circumstances,  a  great 
number  between  them 
are  dead  and  those 
which  have  survived 
are  among  others  which 

are  not  capable  of  de- 
Fig.  26,  —  Various  Stages  in  the   Copulation  of  the        i  ,     ^ 

Ascospores  in  Swcharomyces  Ludwigii.  velopment.    On  account 

of   this    they   will    be 

obliged  to  search  in  other  ascs  for  ascospores  with  which  to  unite. 
They  accomplish  their  union  by  means  of  long  organs. 

This  copulation  is  accompanied  by  numerous  parthenogeneses. 
About  one-fourth  of  the  asco- 
spores germinate  without  under- 
going copulation.  The  analogous 
phenomenon  has  been  found  in 
Willia  Saturnus  and  in  the  yeast 
Johannisberg  II  (Fig.  27),  but 
for  these  two  species  partheno- 
genesis is  still  most  frequent, 
and  half  of  the  ascospores  ger-  Fig.  27. — Various  Stages  in  the  Copulation 
minate  without  copulation.  of  Asc°sP°res  in  Yeast  Johannisberg  II. 

This  second  form  of  copulation  seems  to  be  quite  common  among 
the  yeasts.  H.  Marchand  has  found  this  copulation  in  many  yeasts 
(S.  intermedius,  turbidans,  validus,  vini  Muntzii,  Johannisberg  I, 
S.  Willianus).  In  these  yeasts  about  one-half  of  the  ascospores 
germinate  after  having  fused  two  by  two;  in  Saccharomyces  validus, 
however,  this  copulation  is  accomplished  more  rarely  and  becomes 
exceptional.  Guilliermond  has  observed  the  copulation  of  ascospores 
in  three  yeasts  reported  on  and  secured  from  the  Chevalier  mission 
(Saccharomyces  Mangani,  Lindnerii  and  Chevalieri),  and  also  the 
yeast  from  Pulque  No.  2.  It  has  been  found  by  Kinokotin  in  Sac- 
charomyces paradoxus,  but  in  this  yeast  it  presents  very  special  char- 
acteristics, the  interpretation  of  which  is  rather  close.  Lindner 


SEXUALITY  25 

has  noticed  this  in  another  yeast  which  he  has  not  named.  This  was 
isolated  from  the  mucilaginous  secretions  on  a  tree  in  the  Berlin 
Botanical  Garden. 

Copulation  of  ascospores  does  not  seem  to  be  regarded  as  a  true 
fecundation  but  as  a  phenomenon x  of  parthenogamy  —  a  sexual 
process  replacing  fecundation.  In  fact,  it  may  be  admitted  that  the 
copulation  which  takes  place  in  Schizosaccharomyces,  and  Zygosac- 
charomyces  and  Debaromyces  at  the  moment  when  the  asc  forms 
represents  a  normal  sexual  process  of  yeasts.  The  copulation  which 
takes  place,  then,  among  the  ascospores,  is  a  new  process  and  one 
which  takes  the  place  of  normal  fecundation. 

The  cell  which  gives  rise  to  the  asc  ought  to  be  regarded  as  a 
gamete  developing  parthenogenetically.  As  the  formation  of  ascospores 
necessitates  two  successive  divisions  which  are  not  separated  by 
a  period  of  intercalary  nutrition,  the  nucleus  which  results  is  quite 
devitalized.  This  may  explain  why  the  ascospores  felt  the  need 
of  compensating  for  the  loss  of  chromatin  which  the  nucleus  has 
experienced  in  successive  divisions.  It  is  probable,  however,  from 
what  is  known  with  regard  to  the  higher  ascomycetes,  that  the  asc 
of  the  yeasts  is  the  seat  of  a  reduction  in  the  number  of  chromo- 
somes. The  copulation  of  ascospores  may  intervene  to  replace  the 
fecundation  which  should  occur  at  the  moment  when  the  ascs  are 
formed  and  to  compensate  the  loss  of  chromatin  undergone  in  the 
course  of  mitosis  of  the  asc. 

(C)  Retrogradation  of  Copulation  —  Parthenogenesis 

As  has  been  pointed  out,  in  the  great  majority  of  yeasts,  es- 
pecially those  which  are  of  industrial  significance,  one  does  not 
find  any  trace  of  sexuality.  As  among  species  which  present  a  copu- 
lation at  the  moment  when  the  asc  is  formed,  it  has  become  es- 
tablished from  numerous  cases  of  parthenogenesis  that  the  yeasts 
which  do  not  have  sexuality,  represent  parthenogenetic  forms  derived 
from  primitive  sex  forms.  The  asc,  when  it  has  not  resulted  from 
a  copulation,  has  then  the  import  of  a  gamete  having  developed  by 
parthenogenesis,  that  is,  a  parthenospore.  The  yeasts  may  be  re- 
garded as  a  group  of  fungi  which  have  gone  toward  parthenogenesis 
by  evolution  or  by  force  of  unknown  circumstances,  and  which  are 

1  This  phenomenon  is  comparable  to  that  which  Brauer  has  observed  in  the 
parthenogenesis  of  an  echinoderm,  Artemia  salina.  In  this  organism,  when  fecun- 
dation has  not  taken  place,  there  is  a  fusion  of  a  second  polar  globule  with  the 
egg,  and  this  fusion  takes  the  place  of  fecundation.  Some  phenomena  which 
appear  to  be  similar  have  been  found  since  among  various  fungi  and  protozoa 
and  have  been  grouped  under  the  name  of  parthenogamy. 


26      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 


Fig.  28.— Parthe- 
n 


losing  their  sexuality.     This  opinion  is  supported  by  a  series  of  very 
striking    iacts  which   are   quite   apparent.     A   consideration   of  the 
development  of  various  members  of  this  group  will   give  the   proof 
of  progressive  disappearance  which  sexuality  in  yeasts  has  undergone. 
In  the  Schizosaccharomyces,  which  present  in  this  connection  very 
interesting  characteristics,  copulation  is  illustrated  in  three  varieties: 
Sch.  odosporus,  Sch.  Pombe,  and  Sch.  mellacei.    In 
the  first  copulation  is  almost  universal.    With  the 
other  two,  on  the  contrary,  copulation  is  rather  fre- 
quent and  a  great  number  of  cells  sporulate  without 
copulation.      A  species  of  Schizosaccharomyces  has 
been  transmitted  from  the  laboratory  of  Professor 
Beijerinck  under  the  name  of  Sch.  mellacei,  which 
neticAscsin    did  not  present   any  trace   of  sexuality;   the  asco- 
romyces  go-    SpOres  originated  in  ordinary  cells  which  had  not 
undergone  copulation.     This  yeast,  which  resembled 
Sch.  mellacei  very  closely,  may  have  been  another  variety. 

Among  the  different  species  of  the  genus  Zygosaccharomyces 
numerous  cases  of  parthenogenesis  have  been  observed.  With  De- 
baromyces  globosus,  however,  this  characteristic  is  more  predomi- 
nant and  many  ascs  originate  without  copulation.  These  may  be 
formed  by  ordinary  cells  or  cells  with  long  projections  giving  them 
the  shape  of  dumb-bells.  (Fig.  28.) 

Some  yeasts  have  lost  their  sexual  characteristics  but  have  re- 
tained traces.  Such  is  the  case  with  Schwan- 
niomyces  occidentalis  which  has  been  described 
by  Klocker.  Guilliermond  has  shown  that  at  the 
moment  of  sporulation  in  this  yeast,  the  cells 
destined  to  form  the  ascs  emit  projections  of 
different  length  by  means  of  which  they  try  to 
unite  two  by  two.  But  the  sexual  attraction 
seems  to  disappear;  it  is  only  exceptional  that 
they  join.  These  little  projections,  then,  may  be 
the  remnants  of  an  ancestral  sexuality. 

Since  then,  Ludwig  Rose1  and  Dombrowski2  have  observed  the  same 
characteristics,  one  in  Torulaspora  Delbrucki,  in  a  new  species  related 
to  this  latter  and  in  a  new  yeast  isolated  from  the  mucilaginous 
secretions  from  an  oak  tree,  which  was  provisionally  named  Yeast 
F;  the  other  in  a  milk  yeast,  S.  lactis.  Yeast  F  was  studied  by 

1  Ludwig  Rose.    Beitrage  zur  Kenntniss  der  Organismen  in  Eichenschleimfluss. 
Inaugural  Dissertation,  University  Berlin,  1910. 

2  Dombrowski,  W.     Die  Hefen  in  Milch  und  Milchprodukten.    Cent.  Bakt.  Abt. 
II,  28,  1910 


.29.  —  Formation 

of  Asc  in  Schwan- 
niomyces  occiden- 
talis. 


SEXUALITY 


27 


Fig.  30. 


Formation  of  the  Asc  in 
F"  of  Rose. 


'Yeast 


Guilliermond  and  was  found  to  present  a  series  of  curious  character- 
istics.1 It  forms  only  a  few  ascospores  and  it  looks  as  if  its  sporogenic 
function  is  on  the  verge  of  extinction.  However,  when  the  yeast 
is  placed  in  media  suitable  for  spore  formation,  almost  all  of  the  cells 
put  out  long  projections  by  means  of  which  they  attempt  to  anas- 
tomose two  by  two.  Often  these  do  not  join  together,  as  if  there 
were  an  opposing  force  at  work, 
or  as  if  by  a  loss  of  sexual 
attraction,  these  projections  when 
they  come  in  contact  continue  to 
elongate  and  thus  form  a  net- 
work. (Fig.  30.)  In  quite  a 
number  of  cases  some  may  estab- 
lish a  union  for  anastomosis  by 
means  of  their  projections  and 
adhere  sufficiently  so  that  a 
slight  pressure  does  not  cause 
them  to  break  apart.  The  wall 
which  separates  the  two  cells  never  quite  disappears  and  in  each  case 
fusion  does  not  take  place.  (Fig.  30,  a.) 

Occasionally,  the  projections  from  the  cells  undergo  an  excessive 
elongation,  since,  not  having  accomplished  their  function,  they  form 
a  little  bud  at  their  extremity.  Often  a  cell  may  give  forth  many 

little  projections  at  different  points  on  its 
surface  in  different  directions  and  even 
these  are  capable  of  ramification.  In 
this  manner  very  peculiarly  shaped  cells 
are  secured  which  look  like  amebae. 
Without  much  doubt  Lindner  observed 
analogous  forms  in  the  pellicle  of  cultures 
of  Saccharomyces  Bailii.  (Fig.  31.)  The 
forms  of  copulation,  depicted  by  this 
author,  seem  to  demonstrate  the  exist- 
ence of  a  copulation  in  this  yeast.  The 
ameboid  cells  represent,  then,  unsuccessful  copulation. 

Only  a  certain  number  of  the  cells  which  have  just  been  de- 
scribed, about  28  per  cent,  enter  the  asc  stage.  All  of  the  others  be- 
come degenerate  forms.  The  ascospores,  in  the  number  of  from  1  to 
4,  originate  in  the  body  of  the  cell;  but  they  are  able  to  enter  the 
interior  of  the  projection  which  assumes  a  bulged  form. 

1  Guilliermond,  A.  Sur  la  regression  de  la  sexualite  dans  les  levures.  Soc.  de 
Biol.  70,  1911.  Sur  la  reproduction  de  Debaromyces  globosus  et  sur  quelques 
phenomenes  de  retrogradation  de  la  sexualite"  des  levures.  Comp.  Rend.  Acad. 
des  Sciences,  151,  1911. 


Fig.  31.  —  Ameboid  Forms  of  S. 
Bailii  in  an  Old  Culture  on 
Nutrient  Gelatin.  Some  of 
the  Forms  have  Sporulated 
(after  Lindner). 


28      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 


Fig.  32.  — '  Partheno- 
genetic  Variety  of 
Saccharomyces  Lud- 
wigii. Germina- 
tion of  Ascospores. 


Thus,  these  examples  indicate  that,  in  many  of  the  yeasts,  the 
ascogenous  cells  which  represent  gametes,  develop  by  parthenogenesis, 
fl          preserving,    nevertheless,   a   little  of   their   sexual 
attraction;   this  is  insufficiently  developed  to  insure 
copulation.     These  species  include  the  yeasts  which 
have  completely  lost  all  traces  of  sexuality,  and  in 
which   sexuality  is  definitely  established,  such  as 
the  Saccharomyces  and  the  majority  of  yeasts. 

We  have  seen  that  certain  yeasts,  as  S.  Lud- 
wigii, Johannisberg  II  and  Willia  Saturnus,  after 
having  lost  their  primitive  sexuality,  have  experi- 
enced the  need  of  compensating  this  by  a  parthenog- 
amy which  consists  in  the  nuclear  and  protoplasmic 
fusion  of  ascospores,  two  by  two.  But  even  this 
secondary  sexuality  seems 

00 


to  disappear.  Thus  in  Sac- 
charomyces Ludwigii  about 
one-fourth  of  the  spores 
germinate  without  copula- 
tion. With  regard  to  the 
yeast  Johannisberg  II,  and 
Willia  Saturnus,  parthe- 
nogamyis  observed  in  only 
one-half  of  the  ascospores. 
We  have  had  opportunity 
to  study  a  variety  of  yeast 
Saccharomyces  Ludwigii, 
arising  from  a  culture  of 
Hansen's,  which  did  not 
offer  any  trace  of  parthe- 
nogamy.  The  ascospores 
formed  long  projections 
which  attempted  to  join 
but  never  accomplished 
this  end.  (Fig.  32.) 

All  this  shows  in  an 
exact  manner  that  the 
yeasts  make  one  of  very 
many  examples  of  a  group 
in  which  sexuality  is  in  the 
act  of  retrograding  and  in 
which  one  may  follow  each 
step  in  the  accomplishment  of  this  phenomenon. 


Fig.  33.  —  Scheme  Representing  the  Development 
of  Forms  of  Yeasts. 

1,  Sch.  octosporus  (isogamic);  2,  Zygosaccharomyces  Barker! 
(isogamic);  3,  "Yeast  6"  of  Pearse  and  Barker  (inter- 
mediate forms  between  iso-  and  heterogamy);  4,  Zygo- 
sacch.  chevalieri  (heterogamy);  5,  Nadsonia  (heterogamy 
and  ascs  resulting  from  the  budding  of  eggs) ;  6,  Yeast 
of  Rose  (parthenogamy  with  traces  of  sexual  attraction); 
7,  S.  cerevisiae,  (parthenogenesis);  8,  S.  Ludwigii  (par- 
thenogamy between  spores). 


From  this  point  of 


GERMINATION   OF  ASCOSPORES 


29 


view,  the  yeasts  are  comparable  to  Saprolegniaceae,  in  which  Bary  has 
pointed  out  a  similar  process. 

If  one  glances  over  the  Saccharomyces,  he  will  be  able  to  dis- 
tinguish four  steps  in  the  progressive  evolution  of  sexuality  (Fig. 
33):  firstly,  those  which  have  preserved  ancestral  copulation  in  origin 
of  the  asc  (Schizosaccharomyces,  Zygosaccharomyces  and  Debaromyces 
globosus)',  secondly,  those  which  have  lost  this  characteristic  but 
may  have  kept  traces  of  it  (Schwanniomyces  occidentalis,  Tondaspora 
Delbrucki,  and  the  yeast  of  L.  Rose);  thirdly,  those  which  have  lost 
ancestral  copulation  and  replaced  it  by  a  parthenogamy  between  asco- 
spores (Johannisberg  II,  Willia  Saturnus  and  S.  Ludwigii);  fourthly, 
those  which  have  lost  all  traces  of  copulation  and  have  become  par- 
thenogenetic. 

Germination  of  Ascospores 

When    placed   under    favorable    conditions,    ascospores   germinate 
and    produce    numerous   vegetative 
cells.     The  manner  of   this   is   dif- 
ferent and  depends  upon  the  species. 

First  Example,  Saccharomyces 
cerevisiae  (Fig.  34):  Let  us  start 
this  discussion  with  S.  cerevisiae, 
which  has  been  studied  by  Hansen.1 
In  the  first  phases  of  germination, 
the  ascospores  undergo  a  swelling 
but  the  wall  subsists.  This  swelling 

is  so  great  that  the  ascospores,   by    Fig.  34.  —  Germination  of  Ascospores 

-    ,,  i  •  i     j.i    '       •    in    Saccharomyces    cerevisiae      (Ob- 

means   of   the   pressure   which  they        servations   from  a   Bottcher  Moist 

exert  on  one  another,  give  the  im- 
pression that  the  asc  is  chambered. 
In  fact,  the  walls  of  the  ascospores 
enter  into  intimate  contact,  and  often 
they  fuse  completely  in  such  a  way 
that  there  are  really  walls  in  the  asc 
which  then  becomes  a  cell  with  many 
chambers.  (Fig.  34,  c,  d,  f,  and  g.) 
During  this  time  the  wall  of  the  asc 
becomes  thinner  and  finally  breaks. 
It  acts  as  a  plaited  veil  which  retains 
the  ascospores,  or  is  completely  ab- 
sorbed by  the  ascospores.  Each  ascospore  then  takes  the  form  of 

1  Hansen,  E.  C.  Recherches  sur  la  morphologic  et  physiologic  des  ferments 
alcooliques,  3,  1891. 


Chamber). 

a,  three  ascospores  in  an  asc,  the  wall  of  which 
has  broken;  a'  and  a",  germination  of  these 
three  ascospores;  b,  asc  with  four  asco- 
spores; b',  germination  of  these  four  asco- 
spores; the  wall  of  the  asc  is  broken;  c,  asc 
with  four  ascospores,  three  of  which  are 
visible;  c'  and  c",  germination  of  these  as- 
cospores; in  c'  the  wall  of  the  asc  is  broken,  in 
c"  budding  has  commenced;  d,  asc  with  three 
ascospores;  d  and  d',  germination  of  these 
ascospores;  in  d"  the  wall  of  the  asc  is  broken; 
e,  e',  e",  e'",  e"",  various  stages  in  the  ger- 
mination of  two  ascospores  in  an  asc;  in 
e'""  the  two  ascospores  have  fused  into  a 
single  one  by  absorbing  the  separating  wall; 
/,  /',  /",  g,  g',  g",  germination  of  ascospores 
in  two  ascs;  h,  h',  and  h",  germination  of  two 
ascospores  in  an  asc;  in  h"  the  wall  between 
the  two  ascospores  has  disappeared;  a 
single  cell  is  thus  formed  (after  Hansen). 


30      MORPHOLOGY  AND   DEVELOPMENT  OF  YEASTS 

an  ordinary  vegetative  cell.  It  commences  to  form  a  bud  at  some 
point  on  its  surface  (d,  e,  and  /).  This  bud  generally  appears  after 
the  rupture  or  the  absorption  of  the  wall  of  the  asc,  but  it  may 
appear  on  the  interior  of  the  asc.  Its  appearance  is  soon  followed 

by  the  formation  of  new  buds  which 

(j/  (~^)  r\    /— )  are  formed  at  various  parts  of  the  sur- 

^  p,     ~     ^       £?   (\      face    of    the    ascospore.     During    the 
SQ  <^  (fjS   CD  ^      formation  of  these  buds,  the  ascospores 
^^    remain    united    but    separate   rapidly. 
Finally  these  germinate;  the  ascospores 
swell    up    and  bud  quickly   after   the 
manner  of  a  vegetative  cell. 

Hansen  has  often  observed,  during 
budding,  the  fusion  of  two  ascospores 
in  a  cell.  But  this  fusion,  which  only 
appears  in  an  exceptional  manner,  is 
not  comparable  to  copulation  which 
has  been  described  in  certain  yeasts, 
as  S.  Ludwigii.  It  takes  place  only 
after  the  ascospores  have  commenced 
to  bud,  generally  between  an  ascospore 
Fig.  35.  — Germination  of  Asco-  which  hag  already  formed  many  buds, 
spores  in  Yeast  Johannisberg  II.  ,  ,  .  ,  .  ,  , 

and  an  ascospore  which  is  not  yet  de- 

a,  two  ascospores  germinate  without  fus- 
ing; e,  n,  i  and  P,  copulation  between  veloped.     Hansen    supposed    that   one 

ascospores  not  included  in  the  same  asc.  . 

served  to  nourish  the  other,  and  that 
a  case  of  parasitism  Was  involved. 

In  the  majority  of  yeasts,  notably  in  Saccharomyces  Pastorianus 
and  in  many  of  the  industrial  yeasts,  germination  occurs  in  the  same 
manner  as  in  S.  cerevisiae.  However  in 
certain  species,  germination  of  ascospores 
is  preceded  by  a  copulation  (parthenog- 
amy) ;  this  is  the  case  with  yeasts 
already  mentioned,  as  Johannisberg  II, 
S.  intermedius,  turbidans,  and  ellipsoideus. 
It  will  be  recalled  that  in  this  species  Fig>  36>  _  Germination  of  Asco- 
the  ascospores,  after  swelling  up,  unite  spores  in  Saccharomyces  Lud- 
two  by  two  by  means  of  a  copulation 

J  In  A,  Fusion  of  Three  Ascospores. 

canal.     A  zygospore  is  thus  formed  by 

the  fusion  of  two  ascospores.  Budding  takes  place  at  the  expense  of 
the  copulation  canal.  (Fig.  35.)  It  is  produced  at  some  point  on  its 
surface.  Often  many  buds  appear  simultaneously  at  different  points 
on  the  canal  of  copulation.  Eventually,  it  happens  that  the  buds 
originate  at  the  expense  of  the  ascospores  themselves.  In  the  mean- 


GERMINATION   OF  ASCOSPORES 


31 


time,  about  one-half  of  the  ascospores  undergo  this  copulation;  the 
others  germinate  by  themselves  without  fusion. 

Second  Example,  Saccharomy codes:  The  ascospores  of  the  genus 
Saccharomy codes,  which  has  been  described  for  the  first  time  by 
Hansen  in  Saccharomyces  Ludwigii,  germinate  in  a  somewhat  special- 
ized manner.  As  we  have  seen,  the  ascospores  of  this  yeast  are  almost 
constantly  in  the  number  of  four  in  each  asc.  The  wall  of  the  asc  is 
able  to  break  before  the  germination  to  free  the  ascospores.  More 
often  they  persist  during  the  first  phases. 
Germination  begins  always  by  a  swelling 
of  the  ascospores.  Whether  these  spring 
from  old  or  young  cultures,  has  much  to 
do  with  the  development. 

In  the  first  case,  the  majority  of  as- 
cospores, a  little  swollen,  undergo  a 
copulation  which  has  been  described  in  a 
preceding  paragraph  and  upon  which  we 
shall  not  dwell  at  this  time.  The  asco- 
spores, ordinarily  united  in  ascs  in  which 
the  membrane  is  not  broken,  put  out 
a  little  protuberance  by  means  of  which 
they  unite  two  by  two.  The  middle  wall 
by  which  they  are  separated  rather 
quickly  disappears.  Sometimes  copulation 
takes  place  slowly;  the  protuberances  put 
out  by  each  cell  elongate  and  fuse  at  the  Fig.  37.  —  Germination  of  very 

ends  after  having  gone  along  together  for      Old  Ascospores  in  Saccharo- 
myces Ludwigii. 
some  time.    The  ascospore  then  looks  like 

a  horse-shoe.  (Fig.  37,  a  and  c.)  In  some  cases,  one  sees  the  fusion  of 
three  ascospores  in  the  same  asc  in  a  single  zygospore  (Fig.  36,  A). 
This,  however,  is  very  rare. 

The  copulation  of  the  ascospores  being  incomplete,  the  zygospore 
is  made  up  of  two  enlargements  united  by  a  copulation  canal  in 
which  is  concentrated  the  nucleus  and  protoplasm.  This  commences 
to  germinate  by  a  procedure  intermediate  between  budding  and 
transverse  division.  The  center  of  this  canal  elongates  into  a  little 
germination  tube.  This  tube  perforates  the  wall  of  the  asc  if  it  is 
not  already  absorbed,  until  it  swells  in  its  upper  part.  This  then 
separates  itself  from  the  rest  of  the  germination  tube  by  a  little 
wall  and  a  slight  circular  constriction.  The  cell  formed  in  this  manner 
detaches  itself  from  the  zygospore,  which  forms  new  cells  by  the 
same  procedure.  Sometimes,  the  first  cell  formed  by  the  zygospore, 
without  detaching  itself,  gives  birth  to  one  or  many  more  cells  which 


32      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 

remain  attached  to  one  another,  making  a  chain.  With  rare  excep- 
tions, the  copulation  canal  of  the  zygospore  germinates  always  in 
the  same  way.  The  simultaneous  production  of  many  buds  is  not 
observed  at  many  points  on  the  surface  as  with  the  yeast  Johannis- 
berg  II. 

About  four  ascospores  germinate  alone  without  preliminary 
copulation.  In  this  case,  after  swelling,  they  form  a  new  germinating 
tube  in  which  the  end  is  enlarged  and  take  the  form  of  an  ordinary 
vegetative  cell.  Here  again  germination  takes  place  only  in  a  single 
direction  and  the  ascospore  forms  only  a  single  germinating  tube  at 
a  time. 

The  germination  of  old  ascospores  is  accomplished  in  a  different 
manner.  Hansen  has  observed  that  old  ascospores,  whether  due  to 
humidity  or  dryness,  lose  their  tendency  to  fuse  and  germinate  alone. 
They  develop,  then,  in  a  peculiar  manner.  Each  forms  a  germinat- 
ing tube  which,  in  developing,  produces  a  long  filamentous  form  of 
very  many  cells  superimposed  and  capable  of  ramifying.  It  presents 
something  the  appearance  of  a  mycelium.  Hansen  has  given  the 
name  promycelium  to  this  formation  and  compared  it  to  the  filaments 
which  result  from  the  germination  of  chlamydospores  of  the  Ustila- 
ginales.  Guilliermond  has  verified  this  observation  in  the  germina- 
tion of  ascospores  from  old  cultures  and  his  observations  have  allowed 
an  explanation  of  this  structure,  improperly  called  a  promycelium. 
As  has  been  said  in  the  preceding  paragraph,  the  ascospores  from 
very  old  cultures  find  themselves  obstructed  in  copulation.  A  great 
number  are  dead,  and  the  cells  which  survive  are  often  isolated  and 
surrounded  by  spores  which  are  incapable  of  developing.  On  account 
of  this  they  may  have  to  search  other  ascs  with  which  to  unite. 
They  send  out  long  tubes  more  or  less  branched  which  may  accom- 
plish a  fusion  but  which  more  often  do  not  unite.  In  this  case  the 
tubes  end  up  by  walling  off  cells  which  dissociate  and  take  the  form 
of  vegetative  cells.  Under  such  conditions  the  greater  part  of  the 
ascospores  find  it  necessary  to  germinate  alone.  One  usually  finds 
a  few  which  are  able  to  copulate. 

Let  us  recall  what  we  have  described  in  S.  Ludivigii,  in  which 
the  ascospores  always  germinate  without  preliminary  copulation. 
However,  many  of  them  preserve  their  traces  of  sexual  attraction, 
and  send  out,  in  germinating,  long  protuberances  which  do  not  accom- 
plish anything. 

The  germination  of  the  ascospores  of  S.  Ludwigii  differs  essen- 
tially from  the  other  yeasts,  and  in  this  one  the  ascospores,  copulated 
or  not,  do  not  produce  many  buds  at  various  points  on  the  surface, 
but  germinate  in  a  single  direction  in  which  they  form  a  sort  of 


GERMINATION   OF  ASCOSPORES  33 

germination  tube.  This  separates  the  ascospore  by  a  transverse  wall 
accompanied  by  a  slight  constriction.  In  this  the  germination  of  the 
ascospores  does  not  differ  from  the  method  of  multiplication  of  cells 
in  this  yeast  which,  as  we  have  seen, 

generally  occurs  at  the  end  of  the  cell      a.    «^.  et^     a;^     a"":A., 
by  a  process  intermediary  between  bud-  ^}     ^^      a?*™* 

ding  and  transverse  partition. 

Third  Example,  Willia:  We  have  seen 
that  the  ascospores  of  the  genus  Willia 
present  a  special  form.      In  Willia  ano- 
mala  they  are  hemispherical  and  are  pro- 
vided  with  a  projecting  edge.     (Fig.  38, 
a.)      At    the   moment    of    germination,    Fig.  38.  —  Germination  of  Asco- 
which  has  been  followed  by  Hansen,  the      g°nsen)!  ^^  ^^  (after 
ascospore  swells  and  during  this  its  jut- 
ting out  border  disappears  but   remains   for  some   time   during  the 

early   stages  of   budding.    The  asco- 
A    ©  OD  P&f  spore  eventually  forms   buds  at   dif- 

ferent  points  on  its  surface. 
0  In  Willia  Saturnus  the  ascospores 


are  lemon-shaped  and  are  girdled  with 
a  projecting  ring.      The  wall  of  the 
3      \j   -H-T     Vj     u  X^j  asc    generally    dissolves    before    ger- 


)/       A       mination.      This  begins  by  a  swelling 
^U      during  which  the  projecting  girdle  dis- 
appears  or  remains;    then  a  series  of 

Fig.    39.  —  Germination    of    Asco-  buds  is  produced  at  various  points  on 
spores  m  Wilha  baturnus.  ^ 

the  surface  ot  tne  ascospore.     In  the 

course  of  budding,  the  projecting  thread  disappears.  (Klocker.1) 
A.  In  many  cases  germination  is  preceded,  as  has 
been  stated  in  a  foregoing  paragraph,  by  a  partheno- 
gamic  copulation  of  the  ascospores.  These,  during 
enlargement,  unite  two  by  two  by  means  of  a  copu- 
lation canal.  B.  The  fusion  is  incomplete  and  the  Fig.  40.— Germina- 
,  .  ,  ,.  .  i  i  i  T  tion  of  Ascospores 

zygospore  which  results  germinates  by  budding  on       jn     Debaromyces 

all  points  of  its  surface,  by  preference  on  the  copu-       globosus    (after 
•i    . .  i  IvlocKerJ . 

lation  canal. 

Fourth  Example,  Debaryomyces  globosus  and  Schwanniomyces 
ocddentalis.  Both  of  these  yeasts  have  peculiarly  shaped  ascospores 
in  which  it  is  wise  to  describe  germination.  In  D.  globosus  the 
ascospore  is  round  or  globoid  and  enveloped  in  a  warty  wall.  When 

1  Klocker,  A.  Eine  neue  Saccharomyces  Art  (S.  Saturnus).  Comp.  Rend, 
trav.  lab.  de  Carlsberg,  6,  1903. 


34      MORPHOLOGY  AND   DEVELOPMENT   OF  YEASTS 

it  germinates,  it  undergoes  at  first  a  swelling  during  which  these 
warts  disappear.  (Klocker.1) 

The  ascospores  of  Schw.  occidentalis  have  also  a  warty  wall  and 
are  divided  into  unequal  parts.  The  largest  of  these  possesses  a  pro- 
jection thread.  At  the  time  of  germination,  the  smallest  part  of 
the  ascospore,  that  which  does  not  possess  the 
projecting  portion,  swells,  loses  its  warts,  and 
gives  the  impression  that  the  ascospore  pos- 
sesses two  walls.  The  larger  part,  that  which 
does  not  undergo  an  enlargement,  appears 
clothed  with  an  outer  layer  which  the  asco- 
spore tears  when  it  grows.  (Fig.  41.) 

Fifth  Example,  Saccharomycopsis  guttulatus: 
The  ascospores  of  this  yeast  are  elongated  and 

Fig.  41.-  Germination  of   clothed>   as    has    been    stated>   with   two  mem- 
Ascospores  in  Schwan-  branes,  an  endoplast  and  ectoplast.     According 

"alZdfng  to^K™)!  to  Wilhelmi*  the  germination  begins  with  an 
enlargement  of  the  ascospore,  which  causes  a 

rupture  of  the  ectoplast.  (Fig.  42.)  This  rupture  is  accomplished 
at  one  end  or  on  the  side.  Soon  after  budding  begins  and  proceeds  in 
the  usual  manner.  During  this  budding,  the  ectoplast  becomes 
irregular,  shrivels  up  and  leaves  a  little  attached  to  the  ascospore. 

Sixth  Example,  Monospora  cuspidata  and  Nemato- 
spora  Coryli:  These  two  yeasts  are  also  characterized  by 
ascospores  with  special  shapes.  In  Monospora  cuspidata 
the  germination  has  been  described  by  Metschnikoff.3 
The  ascospores  shaped  like  needles  germinate  laterally 

and  in  a  prolonged  form  with  oval  buds.    These  break  Fig.    42.  —  Asc 

,     !       ,  mSactharomy- 

apart  slowly.  copsis  guttuia. 

In   Nematospora  coryli,  in  which  the  ascospores  are  tus     Showing 

fusiform  and  terminate,  at  one  or  both  ends,  in  a  long  s^res  at  the 

cilium,  the  disappearance  of  this  cilium  is  soon  accom-  Beginning  of 
plished  and  the  ascospore  assumes  the  shape  of  a  short 

.  i  •    i          -M        mi  11  111  The  exosporium  is 

thick  cell.    These  bud  also  at  one  or  both  ends.  ruptured     (after 

Seventh  Example,  Schizosaccharomyces:  With  Sch. 
octosporus  4  the  ascospores  are  able  to  remain  in  the  interior  of  the 

1  Klocker,  A.     Deux  nouveaux  genres  des  Sacch.     Comp.  Rend,  des  trav.  lab. 
de  Carlsberg,  8,  1909. 

2  Wilhelmi,  A.     Beitrage  zur  Kenntniss  des  Sacch.  guttulatus.     Inaug.  Dissert. 
Bern.  Lena,  1898. 

3  Metschnikoff,    E.      Ueber    eine    Sprosspilzkrankheit    der    Daphnien,    Vir- 
chows  Archiv,  96,  1884. 

4  Guilliermond,  A.     Observations  sur  la  germination  des  spores  du  S.  Ludwigii. 
Bull.  Soc.  de  mycol.  de  France,  2,  1903. 


GERMINATION  OF   ASCOSPORES  35 

asc,  but  very  often  the  wall  of  the  asc  is  absorbed  and  the  spores  are 
set  free. 

In  the  latter  case,  they  may  isolate  themselves  or  remain  united. 
At  the  time  of  germination,  they  commence  to  enlarge  and  become 
large  cells  similar  to  those  in  the  vegetative  stage.  During  this  time 
the  wall  of  the  asc,  if  it  exists  up  to  A 

this  time,  breaks  up  and  is  absorbed.      QJY}  (\^prr)    (L^  CQC~C/ 
But  it  often  remains  in  the  state  of  a  U 

veil  during  the  partition  of  the  asco-  Chv--^> 
spores.  The  ascospores  sometimes  re-  \r^ 
main  spherical  and  form  in  the  middle 
a  wall  which  divides  them  into  two 
daughter  cells.  These  become  round  Fig>  43.  -  Germination  of  Asco- 
and  separate.  But  more  often  they  spores  in  Schizosaccharomyces 
elongate.  (Fig.  14,  a.) 

In  Sch.  mellacei  and  Sch.  Pombe  the  ascospores  germinate  after 
the  absorption  of  the  membrane  of  the  asc.  This  absorption  is 
accomplished  very  quickly.  They  enlarge  and  each  gives  rise  to  a 
little  tube  which  soon  divides  and  forms  two  bacilli-like  cells.  Soon 
these,  in  their  turn,  divide  in  the  same  manner 
and  furnish  numerous  generations  of  vegetative 
cells. 

Direct  Germination  of  Ascospores  in  Asc: 
The  investigations  of  Hansen  and  Guilliermond 
have  shown  that  under  certain  conditions  bud- 
ding may  be  suppressed  and  that  the  asco- 
spores, after  becoming  enlarged,  are  susceptible 
Fig.  44.  —  Abnormal  Ger-  '  .  '  >.  .^ 

mination  of  Ascospores  to  germination  without  undergoing  multiphca- 
in  Saccharomyces  Lud-  ^on      ^^{s  produces,  then,  a  curious  shortening 


wzgii,  on  slices  of  carrot.  , 

ot  the  development. 

The   zygospore   resulting  from 

the  fusion  of  two  ascosporea         Hansen  1  has  observed  this  phenomenon  in 

changes  directly  into  an  asc.  x 

Saccharomyces  cerevisiae  and  the  yeast  Johan- 

nisberg  II  in  the  following  manner.  He  placed  some  ascs  of  this 
yeast  in  beer  wort  in  a  Freudenreich  flask.  At  the  end  of  two  hours, 
the  ascospores  enlarged  and  often  copulated.  After  from  three  to  five 
hours,  the  wall  of  the  asc  broke  and  the  ascospores  grew  larger  and 
larger.  He  placed  some  others  in  Freudenreich  flasks  containing  a 
saturated  solution  of  calcium  sulfate.  (It  will  be  seen  further  on 
that  calcium  sulfate  has  the  property  of  arresting  immediate  budding.) 
Under  these  conditions  the  ascospores  are  not  able  to  germinate  by 
budding  and  immediately  go  into  ascs. 

1  Hansen,  E.  C.    Recherches  sur  la  physiologic  et  la  morphologic  des  ferments 
alcooliques.     Comp.  Rend,  des  trav.  lab.  de  Carlsberg,  5,  1902. 


36      MORPHOLOGY  AND  DEVELOPMENT  OF  YEASTS 


Guilliermond 1  accidentally  observed  the  same  phenomenon  in  the 
same  yeast  and  in  others  (S.  Ludwigii,  Willia  Saturnus)  by  a  pro- 
cedure much  more  simple,  by  making  the  yeast  ascospore  germinate 
on  slices  of  carrot.  In  this  nutrient  medium,  the  ascospores  ger- 
minate very  rapidly  and  produce  numerous  generations  of  vegeta- 
tive cells.  But  at  the  end  of  a  few  days, 
the  multiplication  is  arrested,  probably  by 
an  accumulation  of  toxic  substances  which 
may  play  a  r61e  similar  to  the  chalk. 
The  cells  are  then  caused  to  sporulate. 
But  as  the  majority  of  ascospores  ger- 

OTS  O  f  AnX  ^  )  X  niinate  immediately  in  this  medium, 
(j  O  _  ^  ^ff*&  others,  less  vigorous,  do  not  begin  to  ger- 
minate until  the  vegetative  cells  produced 
by  the  germination  of  the  first  begin  to 
sporulate.  Under  these  conditions,  ger- 
mination of  these  tardy  ascospores  is 
without  doubt  restrained  by  the  presence 
of  toxic  substances,  secreted  by  the  vege- 
tative cells.  Thus  they  are  not  able  to 
bud  nor  be  transformed  into  ascs.  With 
S.  Ludwigii,  for  example,  one  may  see  fused  ascospores  which,  with 
an  enlarged  copulation  canal,  produce  new  ascospores  inside.  We  have 
formed,  in  this  way,  two  swellings  connected  by  an  isthmus  and 
resembling  very  closely  an  asc  of  Zygosaccharomyces  or  Schizosac- 
charomyces.  (Fig.  44.)  Sometimes  the  ascospore  attempts  to  ger- 
minate and  produces  a  tube  for  germination  which,  not  being  able 
to  complete  its  development,  enters  the  asc  stage. 

Guilliermond  has  found  the  same  thing  in  Sch.  octosporus.  Here, 
the  ascospores  are  able  to  fuse  two  by  two  and  form  an  egg  which 
soon  is  transformed  into  an  asc.  Often,  they  undergo  one  or  two 
divisions,  the  daughter  cells  fusing  to  produce  new  ascs.  (Fig.  45.) 

This  direct  germination  of  ascospores  in  the  ascs  is  explained 
easily  by  the  fact  that  the  ascospores  have  the  import  of  a  vegetative 
cell.  It  may  be  able  to  sporulate  when  conditions  are  favorable 
and  may  not  have  need  to  undergo  a  preliminary  multiplication. 

1  Guilliermond,  A.  Observations  sur  la  germination  des  spores  du  S.  Lud- 
wigii. Bull.  Soc.  de  mycol.  de  France,  2,  1903. 


Fig.  45.  —  Abnormal  Germina- 
tion of  Ascospores  of  Schi- 
zosaccharomyces  octosporus  on 
slices  of  carrot. 

The  ascospores  fuse  two  by  two,  pro- 
ducing ascs  immediately,  or  soon 
form  cross  walls  producing  cells  to 
make  ascs. 


CHAPTER  II 
CYTOLOGY  OF  YEASTS 

General  Considerations.    Historical 

FOR  many  years  the  cytology  of  yeasts  has  been  concerned  with 
the  nucleus.  Do  yeasts  have  nuclei  like  other  organisms?  Or, 
on  the  contrary,  are  they  deprived  of  a  nucleus  and  therefore  an 
exception?  The  question  of  a  nucleus  in  the  yeasts  has  given  rise  to 
a  great  number  of  reported  investigations  which  allow  contradictory 
conclusions.  Some  authors,  among  others  Dangeard,  Janssens, 
LeBlanc,  Bouin,  etc.,  have  described  bodies  in  yeasts  which  seemed 
to  them  to  be  nuclei;  but  others,  having  noticed  a  great  number  of 
disseminated  particles  in  the  cells,  have  admitted  the  presence  of  a 
"  Diffused  Nucleus."  They  believe  that  the  chromatin  is  more  or 
less  mixed  with  the  protoplasm  of  the  cell  and  sometimes  condensed 
in  the  form  of  colored  grains.  Eischenschitz,  having  noticed  that 
these  grains  were  particularly  abundant  in  the  vacuole,  admitted 
that  this  last  was  a  sort  of  rudimentary  nucleus. 

This  conception  was  specified  by  Wagner1  in  1898  and  again 
by  Wagner  and  Peniston.2  These  authors  described  in  the  yeasts, 
first,  a  vacuole,  vacuole  nucleare,  filled  with  chromatin  particles,  and 
secondly,  a  nucleole  of  homogeneous  appearance,  situated  at  the 
exterior  of  this  vacuole  but  always  close  to  it.  The  whole  of  this 
vacuole  is  filled  with  particles  of  chromatin  and,  according  to  these 
authors,  is  a  rudimentary  nucleus  representing  a  primitive  step  in  the 
phylogenetic  development  of  the  nucleus. 

Guilliermond3  has  given  this  debated  question  of  yeast  structure 
much  study  since  1901.  It  has  been  shown  that  the  interpretation 
of  Wagner  is  inexact,  and  the  yeasts  have  a  structure  identical  with 

1  Wagner,  H.     The  nucleus  of  the  yeast  plant.     Ann.  of  Botany,  12,  1898. 

2  Wagner,  H.,  and  Peniston,  A.     Cytological  observations  on  the  yeast  plant. 
Ann.  of  Botany,  24,  1910. 

3  Guilliermond,   A.     Recherches  sur    la  structure  de  quelques  champignons 
infe"rieurs.    Comp.  Rend.  Acad.  des  Sciences,  133,  1901.    Recherches  histologiques 
sur  la  sporulation  des  levures.     Comp.  Rend.  Acad.  Sci.  133,  1901.     Recherches 
cytologiques   sur  la  levure.     Thesis   for    Doctor   of   Science,  Paris,    1902.     Re- 
marques  sur  differentes  publications  parues  recemment  sur  la  cytologie  des  levures, 
Cent.  Bot.  26,  1910.    Nouvelles  recherches  sur  la  cytologie  des  levures.     Comp. 
Rend.  Acad.  Sci.  150,  1910. 

37 


38 


CYTOLOGY  OF  YEASTS 


that  of  other  fungi  cells  with  a  perfectly  characterized  nucleus. 
These  investigations  have  shown  that  the  body  which  Wagner  took 
for  a  nuclear  vacuole  is  in  reality  another  thing  —  simply  a  vacuole 
with  metachromatic  corpuscles.  In  contradistinction  to  the  nucleus 
of  Wagner,  it  is  not  homogeneous.  The  existence  of  a  nucleus  can- 
not be  doubted.  The  presence  of  it  is  now  admitted. 

Let  us  now  investigate  with  detail  the  different  elements  which 
make  up  the  yeast  cell,  that  is,  the  nucleus,  cytoplasm,  the  elements 
which  it  contains,  and  finally  the  cell  membrane.  Then  let  us 
review  the  phenomena  which  evolution  has  accomplished  in  the  cell 

The  Nucleus 

The  nucleus  is  relatively  large  in  comparison  to  the  cell  (about 
1  n  in  diameter).  It  occupies  a  variable  position,  depending  upon 

the  form  of  the  cell  and  its  stage  of  develop- 
ment. Whatever  its  location,  it  is  often 
closely  associated  with  the  vacuole  which 
encloses  the  metachromatic  granules.  This 
is  easily  explained  by  the  fact  that  the 
nucleus  seems  to  play  a  r61e  in  nutrition  and 
secretion,  and  that  the  vacuole  is  the  seat  of 
an  intense  secretion. 

The  nucleus  almost  always  presents  a 
well-differentiated  structure.  It  is  surrounded 
by  a  colored  membrane,  filled  with  a  colorless 

-  ~  Saccharomyces    interior  in  whjch  are  a  nucleolus  and  a  chro- 
ellipsoideus  with  Its 
Nucleus.  matic  framework  more  or  less  abundant  and 

From    a    Preparation    stained    visible,  depending  upon   the   species.     (Fig. 

with  Hematoxylin.  _  \         T        o  ..  .  .         ,  .•      f 

46.;  In  S*  ceremsiae  this  chromatic  frame- 
work is  very  distinct,  the  chromatin  being  particularly  abundant. 
By  its  structure  this  nucleus  is  not  distinguishable  from  the  nucleus 
of  other  organisms,  notably  those  which  are  present  in  most  of  the 
fungi.  Today  the  nucleus  is  unique  even  in  those  cells  which  are  elon- 
gated and  which  tend  to  form  the  rudiments  of  a  mycelium.  Later 
on  we  shall  take  up  nuclear  division. 

The  Cytoplasm  and  Its  Different  Products 

The  cytoplasm  undergoes,  as  will  be  seen  in  connection  with  the 
evolution  of  cells,  great  variations  in  the  course  of  development. 
Very  dense  and  homogeneous  in  young  cells,  it  encloses  in  the  ma- 
jority of  yeasts,  especially  the  spherical  or  oval  yeasts  (S.  cere- 
visiae,  ellipsoideus,  Pastorianus,  etc.),  a  vacuole  filled  with  corpuscles 


THE  CYTOPLASM  AND   ITS    DIFFERENT   PRODUCTS    39 


Fig.  47.  —  Yeast  Cells  with  Nuclei  (n)  and 
Metachromatic  Corpuscles  (cm).  From  a 
Stained  Preparation. 

1-4,  S.  cerevisiae,  beginning  of  fermentation;  5,  S-  Lud- 
wigii,  beginning  of  fermentation;  6-10,  evolution  of 
metachromatic  corpuscles  during  sporulation. 


(nuclear  vacuole  of  Wagner).  Sometimes  in  the  long  yeast  cells 
(S.  Ludwigii,  Sch.  Pombe  and  mellacei,  Mycoderma)  it  possesses 
two  such  vacuoles  situated  at  both  ends  of  the  cell  and  separated  by 
a  sort  of  very  dense  cytoplasmic  bridge  in  which  the  nucleus  is 
situated.  (Fig.  47,  5.)  In  the  course  of  development  other  vacuoles 
may  appear  at  the  side  of  these  and  include  glycogen,  giving  the  cell 
an  alveolar  appearance.  At  the  same  time,  the  cytoplasmic  struc- 
ture which  limits  these  vacuoles  is  filled  with  numerous  grains  of 
various  forms  and  sizes  which 
are  colored  in  the  same 
manner  as  the  nucleus  which 
we  have  called  "  basophile 
grains."  Finally,  droplets  of 
fat  are  also  often  seen. 

The  cytoplasm  is  then 
the  seat  of  numerous  secre- 
tions: metachromatic 
granules,  glycogen,  basophile 
grains,  and  fats.  The  char- 
acters of  these  special  prod- 
ucts will  now  be  taken  up. 

A.  Metachromatic  Granules:  The  metachromatic  granules,  which 
were  first  studied  by  Guilliermond,1  constitute  the  most  impor- 
tant elements  which  are  found  in  yeasts.  They  seem  to  play  a 
very  important  role  in  cellular  life.  These  bodies  are  almost  ex- 
clusively localized  in  certain  vacuoles,  be  it  in  a  regular  vacuole 
occupying  the  middle  of  the  cell  or  in  two  polar  vacuoles.  They  are 
able  to  exist  also  in  the  cytoplasm  which  surrounds  the  vacuoles. 
It  is  there  that  they  seem  to  originate  elaborated  by  cytoplasm  and 
probably  with  the  participation  of  the  nucleus,  because  it  is  almost 
always  in  contact  with  the  vacuoles.  Once  elaborated  by  the  cyto- 
plasm, they  localize  in  the  vacuoles,  at  whose  expense  they  enlarge, 
in  order  to  eventually  dissolve  at  the  time  of  their  utilization.  •  The 
metachromatic  corpuscles  are  easily  visible  in  living  cells  where  they 
appear  as  refractive  particles  in  the  vacuoles,  and  seem  to  possess  a 
Brownian  movement.  They  may  be  fixed  in  the  living  condition  by 
such  dyes  as  methylene  blue,  neutral  red,  toluidin  blue,  etc. 

1  Guilliermond,  A.  Recherches  sur  la  structure  de  quelques  champignons 
inferieurs.  Comp.  Rend.  Acad.  Sci.  133,  1901. 

Guilliermond,  A.  Recherches  histologiques  sur  la  sporulation  des  levures. 
Comp.  Rend.  Acad.  Sci.  133,  1901. 

Guilliermond,  A.  Recherches  cytologiques  sur  les  levures  et  quelques 
moissures  a  forme  levures.  Thesis  for  the  Doctorate  at  the  Sorbonne,  Storck,  exit. 
Lyon,  1902. 


40  CYTOLOGY  OF  YEASTS 

The  investigations  of  Dangeard  have  shown  that  the  metachro- 
matic  granules  are  produced  from  a  condensation  of  a  metachromatin 
matter  existing  in  the  vacuole  in  the  state  of  a  colloidal  solution. 
In  the  living  cell  they  are  rather  numerous,  but  staining  brings  out 
larger  numbers.  The  metachromatin  precipitates  under  the  in- 
fluence of  the  vital  stains.  It  acts  the  same  way  towards  fixatives. 

After  fixation  by  alcohol,  the  granules  are  stained  more  deeply 
than  the  nucleus  by  the  nuclear  stains.  They  take  the  colors  of  the 
basic  aniline  blue  and  violet  dyes  and  assume  a  color  between  a  red 
and  violet.  With  hemotoxyline  or  hematine  they  are  colored  a  wine 
red.  This  metachromatism,  to  which  they  owe  their  name,  distin- 
guishes closely  between  the  nucleus  and  other  bodies  in  the  cell. 

The  metachromatic  corpuscles  are  present  in  great  abundance 
not  only  in  yeasts  but  also  in  many  of  the  Protista.  We  have  shown 
that  they  are  identical  with  other  bodies  which  have  been  observed 
formerly  in  the  bacteria  and  Cyanophyceae  by  Babes  and  Biitschli,  and 
regarded  as  grains  of  chromatin.  Biitschli  has  called  them  "  Red 
granules  "  on  account  of  their  metachromatism  and  we  have  retained 
by  reason  of  its  priority  the  term  "  metachromatic  granules  "  sug- 
gested by  Babes.  This  ought  to  be  used  also  instead  of  the  term 
"  grains  volutine "  proposed  by  A.  Meyer.1  The  metachromatic 
granules  have  been  pointed  out,  since,  in  the  fungi,  algae,  and 
protozoa.  On  the  contrary  they  do  not  seem  to  be  present  in  the 
Metazoa  or  the  Metaphytes.  According  to  the  observations  of  A.  Meyer 
and  Guilliermond,  in  collaboration  with  Beauverie,2  the  globoid  grains  of 
the  Phanerogames  contain,  associated  with  glycerol  or  saccharine 
phosphates,  a  nitrogenous  substance  which  seems  to  be  much  like 
the  substance  which  makes  up  the  metachromatic  granules.  This  is 
metachromatin,  more  or  less  like  that  which  is  found  in  yeasts.  The 
granulations  of  Mastzellen  or  leucocytes  present  histo-chemical  prop- 
erties, much  like  those  of  metachromatin,  as  has  been  pointed  out 
by  the  investigations  of  Guilliermond3  and  Mawas. 

There  is,  then,  sufficient  evidence  for  considering  the  meta- 
chromatin as  composed  of  nucleic  acids.  The  recent  investigations  of 
van  Herwerden  have  given  good  reasons  for  favoring  this  hypothesis. 
By  cultivating  yeasts  in  media  completely  deprived  of  phosphates, 
this  author  has  noticed  that  these  yeasts  never  contain  the  least 

1  Meyer,    A.      Orientierende  Untersuchungen  iiber  Verbreitung,   Morph.  und 
Chemie  des  Volutins.     Bot.  Zeitg.  62,  1904. 

2  Beauverie,  J.,  and  Guilliermond,  A.     Note  preliminaire  sur  les   globo'ides. 
Comp.  Rend.  Acad.  Sciences,  1906. 

3  Guilliermond,  A.,  and  Mawas,    J.      Caracteres   histo-chimiques   des   granu- 
lations des  Mastzellen.    Comp.  Rend.  Acad.  Biol.  64,  1908. 


THE  CYTOPLASM  AND  ITS  DIFFERENT  PRODUCTS     41 

trace  of  metachromatic  granules  in  their  cells.  On  the  other  hand, 
by  cultivating  these  yeasts  which  have  been  deprived  of  their  granules 
in  media  with  phosphate,  van  Herwerden  has  noticed  the  immediate 
appearance  of  metachromatic  corpuscles.  A  nucleic  acid  compound 
is  extracted,  along  with  volutin  or  metachromatin,  by  dilute  alkali 
from  Torula  monosa  and  Saccharomyces  cerevisiae.  This  cannot  be 
obtained  from  an  equal  quantity  of  volutin-free  culture.  This  seems 
to  prove  what  has  been  indirectly  supported  in  the  past,  that  meta- 
chromatin is  made  up  of  a  nucleic  acid  compound.  No  doubt 
obtains  but  that  the  nucleic  acid  from  yeast  originally  came  from 
the  volutin.  This  nucleic  acid  is  decomposed  by  a  nuclease  formed 
in  the  Torula  cells,  in  which  process  the  formation  of  phosphoric 
acid  could  be  demonstrated.  The  metachromatin  free  cultures  also 
contain  a  nuclease.  This  is  contrary  to  the  opinion  of  Henneberg 
who  claims  that  the  metachromatin  is  the  enzyme  itself.  This  sub- 
stance, according  to  van  Herwerden,  is  probably  a  nucleic  acid  and 
possibly  a  reserve  material.  While  it  may  not  be  indispensable  for 
the  growth  of  the  cells  it  may  be  of  importance  in  their  individual 
development.  It  may  be  related  to  the  fermenting  ability  by  supply- 
ing small  amounts  of  phosphates1  which  may  be  liberated  from  the 
nucleic  acid  by  the  nuclease. 

The  metachromatic  corpuscles  are  certainly  nitrogenous  products, 
but  their  exact  chemical  nature  is  not  completely  known.  However, 
after  .the  investigations  of  Meyer,  Kohl,2  and  Reichnow,3  it  is  ad- 
mitted that  they  result  from  a  combination  of  nucleic  acids.  Kohl 
regards  them  as  nucleoproteins.  Meyer  has  demonstrated  that  the 
histo-chemical  reactions  of  metachromatin  resemble  those  of  nucleic 
acid  and  that  there  are  other  organisms  which  chemical  analysis 
reveals  to  have  more  nucleic  acid,  as  the  yeasts  and  certain  bacteria, 
which  contain  more  chromatin.  Kossel  has  been  able  to  isolate 
from  yeasts  a  large  amount  of  nucleic  acid,  and  this  seems  dispro- 
portionate to  their  relatively  larger  nucleus.  It  is  probable  that  a 
greater  part  of  this  nucleic  acid  comes  from  chromatin.  Reichnow3 
has  demonstrated  that  in  Haematococcus  pluvialis,  which  normally 
contains  much  metachromatin,  this  substance  disappears  and  does 
not  re-form  when  the  alga  is  cultivated  in  a  medium  entirely  devoid 
of  phosphorus.  Nucleic  acid  is  especially  rich  in  phosphorus.  On 
the  other  hand  the  researches  of  Giemsa  seem  to  indicate  that 

1  The  investigations  of  Levene  and  Kossel  have  indicated  the  presence  of 
large  amounts  of  phosphoric  acid  in  yeasts. 

2  Kohl,  G.     Hefepilze,  Leipzig,  1908. 

3  Reichnow,   E.     Untersuchungen  an  Haematococcus  pluvialis.     Arb.  a.  d. 
Kaiserl.  Gesundheitsamte,  33,  1909. 


42  CYTOLOGY  OF  YEASTS 

the  affinity  of  the  nucleus  for  dyes  depends  upon  the  content  of 
metaphosphoric  acid.  Perhaps,  with  a  little  reservation,  we  may 
explain  the  staining  properties  of  the  metachromatin  in  the  same 
manner. 

With  regard  to  the  role  of  the  metachromatin,  our  knowledge  is 
happily  more  complete.  Certain  bacteriologists  have  tried  to  connect 
the  pathogenicity  of  bacteria  with  their  content  of  granules.  They 
have  regarded  these  as  the  toxic  products  of  the  bacteria  or,  more 
definitely,  as  products  initial  to  the  secretion  of  toxins.  This  theory 
was  supported  by  Behring,1  who  pretended  to  have  extracted  the 
metachromatin  from  Bacterium  tuberculosis  and  stated  that  this  sub- 
stance corresponded  to  the  toxin  of  that  Bacterium.  According  to  him 
a  gram  of  this  substance  on  the  dry  basis  will  be  as  toxic  as  a  liter 
of  Koch's  tuberculin.  It  is  probable  that  the  metachromatin  isolated 
by  Behring  was  not  in  the  pure  state.  The  investigations  of  Guillier- 
mond  have  indicated  that  the  metachromatin  has  no  relation  to 
toxins,  and  that  it  ought  to  be  regarded  as  a  reserve  product. .  The 
metachromatic  granules  are  quite  abundant  during  periods  of  great 
vital  activity  in  the  yeasts.  They  diminish  and  finally  disappear  in 
old  cultures.  They  disappear  very  quickly  in  yeasts  undergoing 
inanition. 

Henneberg2  has  attempted  to  show  that  the  metachromatic 
granules  are  related  to  fermentation.  According  to  this  investigator, 
it  is  during  the  period  of  greatest  fermenting  activity  that  these 
bodies  are  most  highly  abundant  in  the  yeast  cell.  This  is  also 
accompanied  with  an  increase  in  metachromatin  (volutin).  The 
addition  of  phosphates,  which  caused  a  great  increase  in  the  ferment- 
ing ability  of  the  cells,  also  caused  an  increase  in  metachromatin. 
Henneberg  thinks  that  metachromatin  is  the  zymase  itself.  Since 
these  metachromatic  corpuscles  are  found  in  all  fungi,  Henneberg 
states  that  metachromatism  may  be  a  general  reaction  for  a  certain 
group  of  enzymes,  just  as  the  guaiac  reaction  is  characteristic  for  all 
oxidases.  This  theory  is  almost  untenable. 

The  role  of  the  metachromatin  explains  itself  when  the  sporula- 
tion  of  yeasts  is  studied.  It  has  been  stated  that  the  metachromatic 
corpuscles  accumulate  in  yeast  cells  destined  to  sporulate.  They 
dissolve,  and  following  this,  are  absorbed  by  the  ascospores,  and 
disappear  entirely  in  the  maturity  of  these  cells.  (Fig.  47.)  They 
undergo  the  same  evolution  as  the  fats  and  glycogen,  which  are 

1  Behring.     Congres  de  la  tuberculose,  Paris,  1906. 

2  Henneberg,  Volutin  of  the  yeast  cell.    Woch.  Brau.  32  (1915),  301-4,  320-3, 
326-9,   335-7,   345-7,  351-4..    On  the  volutin   (metachromatic  granules)   in  the 
yeast  cell.     Cent.  Bakt.  Abt.  II,  45-  (1916),  50-62. 


THE  CYTOPLASM   AND   ITS  DIFFERENT   PRODUCTS    43 

very  abundant  in  the  cells  about  to  sporulate,  and  play,  like  them, 
the  role  of  reserve  material.  The  results  which  have  been  secured 
by  Guilliermond l  on  the  evolution  of  the  metachromatics  in  the 
higher  ascomycetes  and  various  molds  have  confirmed  this  opinion. 
In  the  young  ascs  of  the  higher  ascomycetes,  the  many  met  achro- 
matic granules  collect  about  the  asco- 
spores  in  formation  when  they  are 
finally  absorbed  by  the  ascospores.  In 
the  molds  (Penicillium,  Sterigmato- 
cystis)  they  accumulate  in  the  fruiting 
heads  and  serve  in  the  nutrition  of 
the  conidia.  Van  Herwerden  admits 
that  these  bodies  represent  reserve 

•  •      \V*"*~  {      '  *  ^  ^p% 

products  which    will    be   decomposed 

by  a   nuclease  with  the  formation  of     FiS-  48.  —  Yeast  cells  with  a  for- 

.  .  mation  of  mucilaginous  network. 

phosphoric  acid,  and  this   favors    the        The  network  has  been  obtained 

fermentation.  by  Partial  drving- 

Arna  +  o  2   frr,^   L-inrla    r\t        l>  Part  of   the    cells    has  fallen.     In  2  and 
Amata,     tWO  KindS  OI  3  it  is  seen  that  the  network  may  affect 

p   Hpmnn-          the  form  of  th,e  entire  wall>  for  examPle 
between  a  and  6;  a  is  a  vegetative  cell; 

in    tV«p   vpf»«t«    hv    RniiHan     TTT  &  an  asc  with  two  ascospores;  4  a,  three 

in  tne  yeasts  D>    soudan  111.       cells  living  in  the  network  (according 

Some    resist    the    fat    solvents    after 

treatment  with  organic   acids   and   become  blackened.     The  others 

become  a  brownish  color  after  treatment 
with  organic  acids  and  are  dissolved  in 
xylol  and  ether.  The  first  type  is  less 
abundant  than  the  second  type. 

B.  Glycogen:  Glycogen  was  observed 
for  the  first  time  in  yeasts  by  Errera.3  It 
is  very  abundant  in  the  cells.  It  is  easily 

Fig.  49.  —  Formation  of  Net-  recognized  by  the  brown  color  (mahogany) 
work  and  Yeast  Cells.     The  . J  .  J ' 

Latter    have    been    Colored  which  it   gives    with    iodm    in    potassium 

with  Methyl  Violet  The  iodide.  The  color  disappears  when  the 
Network  is  Uncolored.  Some  .  *T 

Cells  are  still  Found  in  the  solution  is  heated  to  60  ,  but  reappears 
Meshes,  but  Most  of  them  when  it  coolg  Glycogen  exists  in  almost 
are  Removed  (after  Hansen).  . 

all  of  the  yeasts;  however,  certain  species 

do  not  contain  it  at  any  moment  in  their  development,  perhaps  because 
it  is  used  up  as  soon  as  it  is  formed.  In  this  category,  belong  S. 
apiculatus,  exiguuSj  and  the  Schizosaccharomyces.  On  the  contrary,  we 

1  Guilliermond,  A.     Contr.  a  I'Stude  de  la  formation  asques  et  de  l^piplasm 
des  Ascomycetes.    Rev.  ge"n.  de  Bot.  14,  1903. 

2  Amata,  A.     Ueber  die  Lipoide  der  Blastomyceten.    Cent.  Bakt.  Abt.  II,  42, 
1915. 

3  Errera,  L.    L'epiplasm  des  ascomycetes  at  le  glycogene  des  vege"taux.    Thesis 
d'agregation  des  sciences,  Brussels,  1882. 


44 


CYTOLOGY  OF  YEASTS 


have  seen  that  the  ascospores  of  the  Schizosaccharomyces  contain  some 
starch  which  collects  in  the  wall.  This  substance  replaces  glycogen 
and  is  used  as  a  reserve  during  germination  of  the  spores.  Glycogen 
appears  in  the  cells  from  the  beginning  of  fermentation  and  reaches  its 
maximum  after  48  hours.  It  is  almost  always  localized  in  the  vacuoles 
distinct  from  those  which  contain  the  metachromatic  granules. 
It  diminishes  gradually  and  disappears  entirely  towards  the  end  of 
fermentation.  During  sporulation  it  accumulates  in  great  quantities 
in  the  ascs  and  is  absorbed  by  the  ascospores  during  their  maturity. 

C.  Basophile  Granules:    These  granules,  very  rare  in  young  cells, 
become  very  numerous  in  course  of  development,  especially  between 

12  and  24  hours.  (Fig.  50,  5  and  8.) 
They  are  not  visible  in  the  living  cells 
and  do  not  take  stains.  For  the  most 
part,  they  resist  fixation  and  present 
somewhat  the  same  color  characteristics 
as  the  chromatin.  They  are  stained 
especially  by  hematoxylin  which  gives 
them  an  intense  black  color  like  the 
nucleus.  This  is  less  resistant  and 
they  are  easily  decolorized.  With  the 
other  nuclear  stains,  they  differentiate 
themselves  less  closely  from  the  nu- 
cleus. These  granules  offer  variable 
shapes  and  dimensions.  Many  are 
angular,  and  certain  authors,  as  Hiero- 
nymus  and  Kohl,  have  regarded  them  as  crystalloids  of  protein.  A 
close  examination,  however,  reveals  that  they  are  not  crystalline. 

The  basophile  grains  are  probably  albuminoid  substances,  but  it 
is  not  possible  to  state  precisely  their  r61e.  They  are  in  all  cases 
substances  of  nutrition  (reserve  materials)  and  do  not  seem  to  have 
a  relation  to  fermentation,  because  they  appear  as  well  in  yeasts 
cultivated  under  aerobic  conditions,  as  in  yeasts  in  the  process  of 
fermentation.  On  the  other  hand,  they  are  numerous  at  the  moment 
of  sporulation  and  contribute  to  the  formation  of  the  ascospores. 

D.  Fats:    These  are  present  in  the  living  cells  under  the  form  of 
refractive  granules  of  variable  size,   situated  in  the  cytoplasm,  and 
are  stained  brown  with  osmic  acid.     Will   has  been   able  to   color 
them  red  by  means  of  tincture  of  alkanna  and  has  brought  about 
their  dissolution  by  ether.     Very  abundant  at  the  beginning  of  de- 
velopment in  certain  species  (Debar omyces  globosus,  Sch:  occidentalis, 
Torulaspora,  Torula),  these  fat  globules  are  absent  or  rather  widely 
distributed   in  other   yeasts;  especially  the   industrial   varieties.     In 


Fig.  50.  —  Saccharomyces  cerevisiae 
in  a  Preparation  Colored  with 
Ferric  Hematozyline. 

1  to  4,  beginning  of  fermentation;  5  to  8,  be- 
tween 12  and  24  hours;  9,  after  48  hours. 


THE  MEMBRANE  45 

the  majority  of  yeasts,  they  appear  especially  during  sporulation  and 
serve  as  food  for  the  ascospores.  They  are,  then,  reserve  products. 
Fat  globules  are  also  observed  in  old  cells,  but  in  this  case  seem  to  be 
due  to  a  degeneration  of  the  cytoplasm. 

The  Membrane 

The  membrane  of  the  yeasts  is  thick  and  presents  a  double  layer 
quite  distinct.  With  the  exception  of  S.  granulatiLS,  it  is  provided 
with  isolated  granulations  which  are  placed  in  regular  fashion.  We 
have  seen  also  that  the  ascospores  of  certain  yeasts  may  offer  a 
warty  membrane.  The  chemical  constitution  of  the  membrane  is 
only  slightly  known. 

According  to  chemical  analyses  of  Schlossberger,  it  contains  a 
special  cellulose  which  resembles  fungine  or  metacellulose,  and  is 
distinguished  from  the  true  cellulose  by  its  insolubility  in  ammoniacal 
cupric  oxide,  and  reacts  differently  toward  iodin. 

Liebermann  and  Bitto  in  treating  yeasts  successively  with  acids 
and  alkali  obtained  a  cellulose  which  gave  the  zinc  chloride  reaction. 

According  to  Salkowski,  this  cellulose  is  colored  brown  by  iodin. 
Meigen  and  Spreng1  claimed  that  they  did  not  secure  the  true 
cellulose  from  yeast  membrane  but  a  hemicellulose  which  was  easily 
hydrolyzed  by  the  prolonged  action  of  acids  and  alkalis.  On  the 
contrary,  according  to  Will  and  Casagrandi,  the  membrane  was  not 
colored  by  iodin  nor  by  the  ordinary  stains  for  cellulose.  They 
found  no  cellulose.  According  to  Casagrandi,2  pectose  makes  up 
the  membrane.  Mangin 3  believed  that  it  was  composed  of  callose. 
Tan  ret  and  Visselingh  found  chit  in  in  the  membrane  of  beer  yeast. 
Whatever  is  the  case,  the  membrane  stains  blue  with  Ehrlich's  methyl- 
ene  blue  and  Hanstein's  aniline.  This  membrane  is  especially  visible 
in  the  durable  cells  in  which  it  thickens  considerably. 

According  to  Will  and  Casagrandi,  the  membrane  of  the  durable 
cells  is  made  up  of  two  layers.  (Fig.  10.)  When  the  yeast  is  treated 
with  4  or  5  per  cent  hydrochloric  acid,  washed  and  dried,  and  stained 
with  fuchsin  according  to  the  method  of  Strasburger,  the  outer  layer 
takes  a  violet  red  color  which  is  surrounded  by  a  colorless  layer. 

Yeasts  secrete  under  certain  conditions  mucilaginous  substances 
which  collect  the  cells  into  a  sort  of  network  quite  similar  to  zo- 
ogloea.  Hansen  first  attracted  attention  to  this  phenomenon  which 

1  Meigen   and  Spreng.     Ueber  die  Kohlhydrate   der   Hefe.     Zeitschr.   Phys. 
Chemie,  55,  1908. 

2  Casagrandi,  O.    Saccharomyces  ruber.    Ann.  d'Igi  sperim.  7  and  8,  1898. 

3  Mangin,    L.     Observations   sur   la   constitution   de   la   membrane   chez   les 
Champignons.    Comp.  Rend.  Acad.  des  Sciences,  107,  1893. 


46  CYTOLOGY  OF  YEASTS 

appeared  to  play  a  role  in  the  coagulation  of  yeasts,  followed  by  a 
clarification  of  the  liquid.  This  is  comparable  to  the  agglutination 
which  is  found  among  the  bacteria.  Hansen  has  obtained  the  pro- 
duction of  a  mucilaginous  network  by  placing  brewery  yeast  in  a 
covered  bowl  and  letting  it  stand  while  it  slowly  dries.  When 
a  portion  of  this  yeast  was  examined  in  water,  the  formation  of  an 
entangling  network  was  observed.  (Fig.  48.)  Similar  formations  are 
observed  in  yeasts  placed  on  gypsum  blocks  or  on  gelatin.  Hansen 
has  observed  the  same  phenomenon  in  cells  from  scums  yeasts  of 
certain  species.  This  network  is  brought  out  especially  when  the 
cells  are  stained  with  methyl  violet  or  methylene  blue. 

Certain  pathogenic  yeasts  protect  each  cell  by  means  of  a  thick 
capsule  which  is  mucilaginous  in  nature.1  Certain  yeasts  seem 
to  unite  with  one  another  in  a  constant  manner  by  means  of  a 
gelatinous  substance.  Lindau  has  observed  this  in  yeasts  which  he 
has  studied. 


Changes  in  the  Cell  during  Fermentation 

The  structure  of  yeasts  is  easy  to  interpret  at  the  beginning 
of  development.  During  the  active  period  of  fermentation  they  be- 
come more  complex.  What  complicates  the  subject  at  this  moment 
is  a  very  active  secretive  action.  Like  all  secreting  cells,  they  present 
a  series  of  cytological  phenomena  in  connection  with  the  secretions. 

Let  us  observe  these  modifications  which  are  produced  in  the 
cells  in  the  course  of  fermentation  by  taking  S.  cerevisiae  as  an 
example.  At  the  beginning  the  cells  possess  a  cytoplasm  very  dense 
and  homogeneous,  a  nucleus  situated  at  the  side  of  the  cell  and  a 
vacuole  filled  with  metachromatic  corpuscles  which  occupies  the 
center.  (Fig.  47,  1  and  4,  and  Fig.  50,  1  and  4.) 

After  24  hours  of  fermentation  the   cell  undergoes  very  impor- 

1  Agglutination  or  flocculation  of  yeasts  is  a  complex  phenomenon  which  is 
little  known.  It  seems  to  be  brought  about  by  a  change  in  the  constitution  of 
the  membrane  which  becomes  viscous.  It  appears  in  connection  with  the  forma- 
tion of  a  gelatinous  network  described  by  Hansen.  This  is  the  agglutination  to 
which  one  attributes  the  clarification  of  wine  after  fermentation.  Beijerinck 
(Die  Erscheinung  der  Flokenbildung  oder  Agglutination  bei  Alkoholhefen,  Cent. 
Bakt.  20,  1908)  distinguished  autoagglutination,  produced  by  the  yeast  itself, 
and  symbiotic  agglutination,  brought  about  by  bacteria  developing  at  the  same 
temperature  as  the  yeasts  (especially  by  the  Leuconostoc  agglutinans).  Agglutina- 
tion is  brought  about  by  the  addition  of  sulfuric  acid,  boric  acid  or  1  per  cent 
of  other  acids.  Microscopically,  the  cells  do  not  show  any  alteration  during  the 
agglutination.  Agglutination  seems  to  be  related  to  the  life  of  the  cells,  for  dead 
yeasts  do  not  agglutinate.  Van  Laer  has  shown  that  borax  causes  the  agglutina- 
tion of  the  yeasts  killed  by  heat. 


CYTOLOGICAL   PHENOMENA  47 

tant  modifications.  The  cytoplasm  is  hollowed  out  by  a  certain 
number  of  little  vacuoles  which  are  distinct  from  the  vacuole  con- 
taining the  metachromatic  corpuscles.  The  cytoplasm,  then,  takes 
an  alveolar  structure.  The  nucleus  always  takes  its  place  at  the 
center;  it  seems  to  swell  and  take  on  an  ameboid  shape.  One  ob- 
serves at  this  time  in  all  of  the  cytoplasm,  and  especially  about  the 
nucleus  and  along  the  walls,  a  large  number  of  basophile  granules 
of  irregular  form,  some  angular  and  others  filamentous. 

After  48  hours  fermentation,  the  glycogenic  vacuoles  fuse  into  a 
large  vacuole  which  takes  up  almost  all  of  the  cell  and  absorbs  the 
nucleus  cytoplasm  and  the  vacuole  with  the  metachromatic  cor- 
puscles. The  cell  is  then  transformed  into  a  sort  of  glycogenic  sac. 
At  this  moment  the  glycogen  seems  to  be  retained  by  the  cell,  for 
it  is  not  consumed.  The  income  is  greater  than  the  expenditure. 
The  basophile  granules  decrease  in  number  and  adhere  to  the  wall 
of  the  cell.  At  this  time,  there  appear  in  the  glycogenic  vacuole  a 
considerable  number  of  small  granules  which  differ  from  the  basophile 
grains  by  their  smaller  dimensions  and  lesser  pigmentation  and  whose 
functions  are  unknown.  At  this  stage  the  nucleus  undergoes  a 
variation  in  pigmentation  which  is  very  close;  it  stains  intensely 
and  takes  on  a  homogeneous  aspect.  At  the  end  of  the  fermentation, 
the  cells  assume  the  structure  which  they  had  at  the  beginning. 

These  are  the  modifications  through  which  yeasts  pass  in  the 
course  of  fermentation:  change  in  the  structure  of  the  cytoplasm, 
appearance  of  grains  of  secretion,  variation  in  pigmentation  of  the 
nucleus  are  the  well-known  phenomena  in  secreting  cells.  For  the 
most  part  the  yeasts  fit  this  scheme  with  a  few  differences  in  detail. 


Cytological  Phenomena  during  Vegetative  Multiplication 

A.  Budding:  We  have  already  described  budding  and  it  will 
not  be  necessary  to  recapitulate  at  this  time.  Let  it  .suffice  simply 
to  indicate  the  cytological  phenomena  which  take  place  during  this 
change.  By  its  appearance,  the  bud  is  made  up  of  a  very  dense 
cytoplasm  containing  a  few  basophile  grams  which  have  emigrated 
from  the  mother  cell.  When  it  has  acquired  a  certain  dimension, 
a  little  vacuole  appears  in  the  midst  of  the  cytoplasm  which  is  filled 
with  metachromatic  corpuscles.  This  vacuole  results  often  from  the 
entrance  of  a  little  of  the  vacuole  from  the  mother  cell. 

During  this  phenomenon,  the  nucleus  occupies  its  usual  position 
even  if  it  is  at  the  opposite  end  from  the  bud,  and  undergoes  no 
modification  until  this  has  acquired  its  definitive  dimension.  At 


48  CYTOLOGY  OF  YEASTS 

this  moment  only,  the  nucleus,  without  changing  its  location,  elongates 
and  takes  the  shape  of  a  dumb-bell.  One  of  the  heads  of  this  enters 
the  bud,  (Figs.  46  and  47.)  A  separation  then  takes  place  which 
frees  the  two  heads  from  one  another.  One  part  remains  in  the 
mother  cell  while  the  other  is  in  the  bud.  Both  nuclei  thus  formed 
retain  for  some  time  the  shape  of  a  club  before  assuming  their  normal 
appearance.  The  nuclear  division  does  not  offer  the  characteristics  of 
karyokinesis  contrary  to  the  opinion  of  other  authors  (Swellengrebel1 
and  Fuhrmann).  It  seems  to  consist  simply  of  a  direct  division. 

B.  Transverse  Division:  Division  is  not  observed  as  we  have  seen 
in  the  Schizosaccharomyces.  It  consists  simply  in  the  formation  in 
the  middle  of  the  cell  of  a  transverse  partition  which  separates  the 
two  daughter  cells.  During  this  phenomenon,  one  may  observe  at 
both  extremities  of  the  cell,  the  formation  of  a  little  vacuole,  since 
the  nucleus  situated  in  the  center  elongates  into  a  dumb-bell,  both 
heads  of  which  are  placed  at  ends  of  the  cell.  The  middle  connecting 
link  is  severed  and  the  two  heads  form  the  nuclei  of  the  two  daughter 
cells  which  are  to  be  separated  by  a  transverse  wall. 

Cytological  Phenomena  of  Sporulation 

We  have  seen  in  the  preceding  chapter  that  a  certain  number 
of  the  yeasts  possess  sexual  processes  which  function  either  at  the 
moment  of  sporulation  or  germination  of  the  ascospores.  In  the  first 
case  (Schizosaccharomyces,  Zygosaccharomyces,  Debaromyces)  the  asc 
results  from  the  isogamic  copulation  of  two  cells.  In  the  second 
(Saccharomy codes,  Willia  saturnus)  copulation  is  effected  between 
two  ascospores  at  the  time  of  their  germination.  We  have  been 
able,  in  order  to  present  clearly,  to  describe  by  anticipation  the 
phenomena  (nuclear  and  cytoplasmic  fusion)  which  take  place  during 
this  copulation.  We  shall  not  repeat  here. 

With  the  exception  of  these  species,  none  of  the  yeasts  present 
any  trace  of  sexuality;  with  them  the  asc  forms  at  the  expense  of  each 
cell  without  preliminary  copulation. 

It  has  been  stated  that  the  sporangium  of  yeasts  is  similar  to 
the  asc  of  the  Ascomycetes,  especially  that  of  Exoascees.  However, 
a  difference  exists  between  the  asc  of  yeasts  arid  the  organ  of  the 
same  name  in  the  Ascomycetes.  In  all  of  the  Ascomycetes  which 
do  not  copulate  at  the  moment  when  the  asc  forms,  especially  the 
Exoascees,  this  organ  includes  by  its  origin  two  nuclei,  and  it  is  only 
after  the  fusion  of  these  two  nuclei  that  it  assumes  its  definite  volume 

1  Swellengrebel,  H.  La  Division  nucteaire  dans  les  levures  pressees.  Ann. 
Institut  Pasteur,  19,  1905. 


CYTOLOGICAL  PHENOMENA  OF  SPORULATION       49 

and  forms  ascospores.  From  recent  studies  l  it  has  been  shown  that 
in  the  formation  of  the  asc,  especially  in  the  Exoascees,  which  constitute 
a  family  of  the  Ascomycetes  very  close  to  the  yeasts,  this  fusion  is 
wanting. 

In  many  yeasts  which  do  not  represent  sexuality,  and  among 
others  in  Saccharomyces  cerevisiae,  Janssens  and  Leblanc2  considered 
that  they  observed  a  nuclear  fusion  in  the  cells  which  were  about  to 
form  ascs  and  considered  this  as  a  sexual  act.  According  to  these 
authors  the  nucleus  of  these  cells  undergoes  a  division  since  both 
nuclei,  which  result  after  being  separated  for  some  time,  blend  together 
into  a  single  nucleus  which  by  successive  divisions  furnishes  the  nuclei 
for  the  ascospores.  But  Guilliermond 3  has  shown  that  this  obser- 
vation is  inaccurate  and  that  one  is  not  able  to  verify  any  nuclear  fusion 
in  the  yeast  cells  which  are  destined  to  sporulate  without  preliminary 
copulation.  Thus  karyogamy  preceding  sporulation  is  lacking  in  the 
yeasts,  a  fact  which  seems  definitely  established  today. 

We  shall  stop  now  to  observe  the  processes  which  take  place  in 
the  cell  when  it  transforms  into  ascs  —  the  division  of  the  nucleus 
and  the  formation  of  ascospores. 

Let  us  take  S.  Ludwigii  as  the  example.  In  this  yeast,  sexuality 
before  the  formation  of  the  asc  has  not  been  observed.  As  we  have 
said,  the  cells  pass  directly  into  ascs  without  preliminary  copulation 
and  this  phenomenon  is  replaced  by  a  fusion  (parthenogamy)  of  the 
ascospores  at  the  moment  of  germination. 

The  cells  which  are  preparing  to  sporulate  assume  a  very  complex 
structure.  They  possess  an  alveolar  cytoplasm  in  which  the  network 
which  surrounds  the  alveoli  shows  inclusions  of  fat  and  numerous 
basophile  granules.  Two  sorts  of  alveoli  may  be  distinguished: 
some  are  filled  with  a  considerable  quantity  of  metachromatic  cor- 
puscles; others  with  glycogen.  The  nucleus  is  placed  at  the  side  "of 
the  cell.  It  surrounds  itself  with  a  thin  layer  of  very  dense  zone  of 
protoplasm  (plasma  sporogenic)  at  the  expense  of  which  the  asco- 
spores are  built  up.  In  this  sporogenic  plasma,  the  greater  portion 
of  the  basophile  grains  accumulate.  (Figs.  47,  a,  and  51.)  All  the 
rest  of  the  cytoplasm,  which  possesses  an  alveolar  structure,  will  not 
be  used  in  the  formation  of  ascospores,  but  will  form  the  epiplasm, 
the  plasma  which  will  be  absorbed  by  the  ascospores  during  their 

1  Dangeard,  who  observed  this  nuclear  fusion   first,    regarded  it   as  a  true 
fecundation.    The  explanation  of  this  phenomenon  is  not  completely  elucidated  and 
remains  obscure. 

2  Janssens,   F.,  and  LeBlanc,   A.     Recherches  cytologiques  sur  la  cellule  de 
levure.    La  cellule,  14,  1898.    A  propos  du  noyau  de  la  levure.     La  cellule,  20,  1903. 

3  Guilliermond,  A.    Le  noyau  de  la  levure.    Annales  mycologici,  2,  1904. 


50  CYTOLOGY   OF   YEASTS 

maturation  and  serve  them  as  food.  At  this  stage,  which  corresponds 
to  the  beginning  of  nuclear  division,  some  important  modifications 
appear  in  the  alveoli  which  contain  metachromatic  corpuscles.  These 
bodies,  increased  in  number  and  diminished  in  volume,  undergo  a  sort 
of  pulverization  which  reduces  them  to  infinitely  small  particles 
which,  in  their  turn,  dissolve,  the  alveoli  taking  the  same  color 
that  pertained  to  the  corpuscles.  This  phenomenon  of  dissolution 
of  the  metachromatic  granules  is  followed  by  the  formation  of  the 
ascospores. 

During  this  time  the  nucleus  undergoes  its  first  division;  but, 
the  very  chromophile  cytoplasm,  filled  with  basophile  grains  which 

surround  it,  does  not  allow  the  divi- 
sion to  be  followed  nor  any  knowl- 
edge with  regard  to  how  it  operates. 
One  is  able  to  see  only  two  small 
nuclei  closely  related  to  each  other 
and  situated  in  a  zone  of  sporogenic 
plasma,  which  advance  by  steps  to 
a  single  large  nucleus.  However, 

Fig.  51.  — Formation  of  Ascospores  in     certain  aspects  of  the  phenomenon 
o.  Ludwigii.  . 

seem   to  indicate  that  the  nucleus 

divides  by  karyokinesis.  This  has  been  put  in  evidence  for  Schizo- 
saccharomyces  octosporus. 

The  two  daughter  nuclei  soon  emigrate  to  both  poles  of  the  cell. 
They  are  followed  by  the  sporogenic  plasma  which  divides  between 
the  two  poles  in  order  to  surround  the  nuclei.  At  this  moment, 
one  may  observe  the  steps  in  which  there  are  two  nuclei  at  each 
end  of  the  cell  surrounded  by  a  zone  of  sporoplasm  and  separated  by 
a  portion  of  the  same  material.  Some  authors  (Janssens  and  Le- 
blaftc)  have  attributed  this  to  sort  of  karyokinetic  structures,  re- 
garding the  two  nuclei  surrounded  by  sporoplasm  as  the  anaphase 
plates  and  the  thread  plasma  which  unites  them  as  an  achromatic 
spindle. 

Soon  the  thread  which  unites  the  two  masses  of  protoplasm  dis- 
appears and  each  nucleus  undergoes  a  division.  We  have  seen, 
then,  the  stages  in  which  two  .small  nuclei  are  placed  one  at  each 
pole  of  the  cell  with  a  mass  of  sporoplasm  about  each.  (Figs.  47, 
7,  and  51.)  The  sporoplasm  concentrates  about  each  and  forms  4 
little  balls  provided  with  a  nucleus  and  placed  in  pairs  at  each  pole. 
These  are  the  ascospores.  These  increase  in  size  and  form  a  mem- 
brane about  them.  At  this  moment  the  epiplasm  becomes  disor- 
ganized and  is  reduced  to  an  alveolar  fluid  containing  fats,  glycogen 
and  metachromatic  corpuscles.  The  metachromatic  granules  slowly 


CYTOLOGICAL   PHENOMENA  OF  SPORULATION        51 

disintegrate  and  are  finally  ingested  by  the  ascospores.  Little  by 
little  they  disappear  entirely,  being  absorbed  by  the  ascospores  during 
their  development.  (Fig.  47,  10.)  The  glycogen  and  the  fats  undergo 
the  same  fate  and  are  absorbed  by  the  ascospores.  A  part  of  these 
different  products  is  consumed  by  the  ascospores,  the  other  is  kept 
in  reserve  in  the  ascospores  to  serve  during  germination.  The  asco- 
spores increase  little  by  little  in  size  and  eventually  occupy  the  entire 
volume  of  the  asc,  after  having  absorbed  the  entire  epiplasm  with 
its  metachromatic  corpuscles,  fats,  and  glycogen. 

The  ascospores  reach  the  adult  state  presenting  a  thick  membrane 
and  a  central  nucleus  from  which  the  cytoplasmic  rays  containing 
fats  start,  and  which  delimit  small  vacuoles.  These  include  a  little 
glycogen  and  a  few  corpuscles,  products  which  will  be  consumed  at 
the  moment  of  germination. 

In  all  of  the  yeasts,  the  cytological  phenomena  are  the  same 
differing  only  in  details.  In  Saccharomyces  cerevisiae,  ellipsoideus,  and 
Pastorianus,  instead  of  forming  at  two  poles,  the  ascospores  originate 
generally  in  the  middle  of  the  cell  in  a  zone  of  sporoplasm.  They 
undergo  one  or  two  divisions  depending  on  the  number  of  ascospores, 
and  the  nuclei  which  result  remain  very  close  to  each  other  in  the 
same  zone  of  sporoplasm  which  soon  concentrates  about  each  of  them 
to  make  up  the  ascospores. 

In  the  Schizosaccharomyces  and  especially  in  Sch.  octosporus,  the 
asc  contains  many  less  metachromatic  corpuscles  and  basophile 
grains  than  in  other  yeasts;  also  the  cytological  phenomenon  of  the 
formation  of  the  ascospores  is  much  easier  to  observe.  After  the 
nuclear  fusion,  the  nucleus  grows  and  soon  undergoes  a  first  division. 
Guilliermond  1  has  shown  that  this  is  a  karyokinesis  similar  to  that 
described  among  the  Ascomycetes.  It  is  almost  always  accomplished 
in  the  direction  of  the  long  axis  and  is  manifested  by  the  presence 
of  an  achromatic  spindle  made  up  of  small  granular  particles  more 
or  less  distinct  which  represent  the  chromosomes  of  the  equatorial 
plate.  The  chromosomes  then  distribute  themselves  along  the  spindle. 
At  this  moment,  the  nuclear  membrane  seems  to  be  absorbed  while 
the  spindle  elongates  in  such  a  manner  as  to  form  granular  masses 
at  each  end  of  the  cell.  The  nucleolus  persists  at  the  side  of  the 
spindle,  but  finally  disappears.  Two  nuclei  are  thus  formed  which 
go  to.  different  parts  of  the  cell.  The  two  daughter  nuclei  which 
result  emigrate  to  both  extremities  of  the  cell  to  undergo  another 
-division  and  sometimes  a  third,  from  which  result  4  or  8  ascospores. 
The  nuclei  thus  formed  are  disseminated  in  the  cytoplasm,  which  has 

1  Guilliermond,  A.  Sur  la  division  nucleaire  des  levures.  Annales  de  Tlnsti- 
tut  Pasteur,  31  (1917). 


52  CYTOLOGY   OF  YEASTS 

an  alveolar  structure.     Each  of  these  surrounds  itself  with  a  small 
zone  of  dense  protoplasm,  and  then  transforms  itself  into  ascospores. 

..       These  grow  at  the  expense  of  the  cyto- 
Qr|     plasm  until  they  occupy  the  whole  asc.1 
The  cytological  phenomenon  of  the 
Fig'  5sP»e?rJn±Ll  ^    formation  of  ascospores  presents  many 

characteristics    in    common   with   that 

observed  in  the  ascs  of  other  Ascomycetes,  especially  the  endomycetes. 
The  germination  of    ascospores  when  they  are  not  accompanied 
by  a  copulation,  does  not  offer  any  special  characteristic.     The  asco- 
spores in  time  swell  up  and  are  transformed  into  vegetative  cells  which 
bud  after  the  normal  procedure.     (Fig.  52.) 

1  Beauverie  has  proposed  a  method  for  staining  ascospores.  The  yeast  should 
be  fixed  in  alcohol  or  formol  and  stained  with  carbol  fuchsin  heating  to  the  point 
where  vapor  is  given  off.  It  should  then  be  decolorized  by  1-3  acetic  acid, 
washed  in  water  and  stained  by  thionine.  The  spores  will  be  stained  red  and 
the  rest  of  the  cell  blue.  This  ability  to  resist  acids  is  especially  marked  in 
Schizosaccharomyces  octosporus.  Beauverie,  J.,  Quelques  proprietes  des  asco- 
spores de  levures.  Technique  pour  leur  differentiation.  Comp.  Rend.  Soc.  Biol. 
80  (1917),  5. 


CHAPTER  III1 

PHYSIOLOGY    OF   YEASTS.     NUTRITION,    RESPIRATION, 
AND    ALCOHOLIC    FERMENTATION 

"W  7~EASTS  are  able  to  undergo  two  very  different  kinds  of  life. 

|  Sometimes  they  live  in  contact  with  air  and  respire  —  under 
aerobic  conditions;  at  other  times,  in  the  absence  of  air.  In 
this  latter  case,  they  take  the  energy  which  is  necessary  from  another 
process.  They  transform  the  greater  portion  of  sugar  at  their  dis- 
posal into  alcohol  and  carbonic  acid.  They  thus  induce  an  alcoholic 
fermentation,  which  is  then  anaerobic.  One  must  distinguish  be- 
tween the  yeast  plant  which  acts  like  other  ordinary  plants  and  the 
yeast  ferment  which  is  the  agent  of  alcoholic  fermentation. 

We  shall  take  up  in  this  chapter  the  general  nutritive  processes 
of  the  yeast,  that  is,  its  nutrition,  respiration,  and  alcoholic  fermenta- 
tion, reserving  for  a  following  chapter  the  study  of  the  relation  of 
yeasts  to  their  external  environment,  of  the  conditions  which  determine 
their  multiplication,  sporulation,  and  parasitism. 

We  shall  begin  by  investigating  the  chemical  make-up  of  the 
yeasts  and  by  studying  the  various  enzymes  which  prepare  foods  for 
absorption. 

GENERAL  PHENOMENA  OF  NUTRITION  OF 
THE  YEASTS 

Chemical  Composition  of  the  Yeasts 

The  different  analyses  of  yeasts  undertaken  by  various  authors 
have  given  variable  proportions  of  C,  H,  N,  O,  and  S.  (Dumas, 
Schlossberger,  Mitscherlich.)  Analysis  of  the  ash  of  yeasts  has  given 
equally  inconstant  results.  Phosphoric  acid,  silicic  acid,  carbonic 
acid,  sulfuric  acid,  hydrochloric  acid,  potassium,  sodium,  sulfur, 
magnesium,  calcium  and  ferric  oxide  and  manganic  oxide  have  all 
been  found  to  exist  in  different  proportions. 

Thus,  as  we  have  seen  in  the  preceding  chapter,  the  yeast  cell  is 
composed  essentially  of  a  membrane  which  seems  to  be  made  up  of 

1  In  the  preparation  of  this  chapter,  the  obliging  collaboration  of  M.  A. 
Polecard,  D.Sc.,  Chief  of  the  Department  of  Physiology  of  the  College  of  Medi- 
cine of  the  University  of  Lyon,  is  acknowledged. 

53 


54  PHYSIOLOGY  OF  YEASTS 

cellulose  or  a  closely  related  substance,  of  an  albuminous  protoplasm 
and  a  nucleus  rich  in  nuclein.  The  yeasts  contain  hydrocarbons, 
albuminoids,  and  fatty  bodies. 

Let  us  consider  successively  these  groups  of  substances. 

Hydrocarbon  Materials:  The  analysis  of  Schutzemberger  has 
shown  the  presence  of  a  substance  like  cellulose  in  the  membrane, 
which  seems  to  be  formed  from  sugar,  and  of  a  gummy  substance 
which  seems  to  be  transformed  from  this  cellulose  under  the  in- 
fluence of  the  chemical  agents  used  in  its  preparation.  Finally 
Errera  and  Clautriau  1  have  disclosed  the  presence  of  glycogen  which, 
according  to  Laurent,  is  able  to  reach  a  concentration  of  32  to  38 
per  cent. 

Fatty  Bodies:  Fats  to  the  extent  of  5  per  cent  of  the  dry  material 
have  been  reported.  However,  in  old  cells,  the  fat  may  increase 
to  even  20  per  cent.  This  is  not  surprising,  for  we  have  stated  that 
it  may  exist  in  two  forms:  one  as  reserve  products  formed,  without 
doubt,  from  the  sugars  (Pasteur);  the  other  seems  to  come  from  a 
protoplasmic  degeneration.  The  first  is  generally  rare  during  fermen- 
tation and  appears  especially  during  sporulation.  The  other  is  observed 
in  old  cells  in  the  state  of  degeneration. 

The  fatty  materials  of  yeasts  are  generally  acid  in  reaction  and 
composed  of  ordinary  fats,  cholesterol,  lecithin  and  phytosterol. 
The  weight  of  cholesterol  may  reach  0.06  per  cent  of  the  dry  yeast, 
according  to  Lowe,  but  increases  in  old  cells.  Hinsberg  and  Ross 
have  pointed  out  the  presence  of  an  ethereal  oil,  not  saponifiable, 
with  a  hyacinth  odor  to  which  he  attributes  the  special  odor  of  yeasts. 

Welter2  in  discussing  a  yeast  which  contains  50  per  cent  of 
protein  states  that  it  contains  4  per  cent  of  fat  and  this  may  be 
increased  up  to  17  per  cent.  It  is  thought  that  the  fat  is  produced 
by  a  transformation  of  sugar.  Bokorny3  states  that  to  secure 
abnormal  fat  formation  in  yeasts,  they  must  be  fed  quantities  of 
carbohydrates  and  proteins.  From  the  data  which  he  secured  he 
regards  the  cell  protein  as  the  source  of  the  fat.  Neuss 4  reports 
a  yeast  which  contained  18  per  cent  of  fat  on  the  dry  basis.  Under 
special  conditions  of  cultivation  this  could  be  increased  to  50  per 
cent.  The  fat  was  similar  to  olive  oil. 

1  Clautriau,  G.     fitudes  chim.  du  glycogene  chez  les  champ,  et  les  levures. 
Ac.  roy.  de  Belgique,  3,  1895. 

2  Welter,  A.    Yeast  fat,  a  new' source  of  fat.     Seifenfabriken,  35,  845-6,  1915. 
Chem.  Abstracts,  10,  124,  1916. 

3  Bokorny,   T.      Yeast    fat.     Allgem.    Brau-Hopfen  Ztg.    55,    1803-5,    1915. 
Chem.  Abstracts,  10,  798,  1916.     Assimilation  of  fat  in  plant  cells,  especially  in 
yeasts.     Arch.  Physiol.  1915,  305-49.     Chem.  Abstracts,  11,  2218,  1917. 

4  Neuss,  O.    Yeast  fat.    Seifenfabr.  36,  38,  1916.    Chem.  Absts.  10,  977,  1916. 


GENERAL  PHENOMENA   OF  NUTRITION  55 

Amato l  demonstrated  the  presence  of  lipoids  in  Saccharomyces 
cerevisiae  by  chemical  and  microchemical  methods.  He  treated  yeasts 
in  fixed  preparations  with  osmic  acid,  finding  that  most  of  the  granules 
became  brown  in  color.  By  their  reaction  to  fat  solvents,  the  majority 
of  these  granules  were  regarded  as  belonging  to  Bernard  and  Bigart's 
labile  fats.  Amato  thinks  that  most  of  the  lipoids  in  yeast  are 
lecithins.  Extraction  of  both  the  washed  and  dried  yeast  with 
ether  gave  a  residue  which,  after  combustion,  and  extraction  with 
sodium  carbonate,  yielded  the  characteristic  phosphoric  acid  pre- 
cipitate with  ammonium  molybdate.  Bokorny2  has  reviewed  this 
subject  from  the  point  of  using  this  yeast  fat  commercially.  The 
need  for  these  fats  was  especially  emphasized  in  Germany  during 
the  war  on  account  of  the  successful  blockade  of  German  ports  by 
the  Allies.  Bokorny  stated  that  much  study  was  required  before  the 
yeast  fat  could  be  put  on  a  commercial  basis.  Neville 3  studied  yeast 
fats  and  found  that  the  principal  fatty -acids  had  the  empirical  formula 
Ci5H3oO2,  but  that  arachidic  acid,  C2oH40O2,  melting  at  77°  C.,  was 
not  very  abundant.  Unsaturated  acids  could  not  be  separated  in  the 
pure  state.  Oxidation  with  KMn04  yielded  the  corresponding  di- 
and  tetrahydroxy  acids  which  indicated  the  presence  of  CieHaoO^, 
Ci8H34O2  and  C18H32O2  in  the  fat.  Cholesterol  melting  at  145-147°  C. 
was  obtained.  Bokorny4  has  made  further  observations  on  yeast 
fat  and  found  a  greater  accumulation  when  the  source  of  nitrogen 
was  peptone  than  when  amino  acids  (glutamic  and  aspartic)  were 
used.  Dilute  urine  to  which  sugar  had  been  added  represented  the 
cheapest  source  of  nitrogen. 

Albuminoids  :  A  material  approaching  egg-albumin  in  characteris- 
tics has  been  found  in  yeasts.  According  to  Trommsdorf  and  Meisen- 
heimer  6  the  cake  obtained  by  the  compression  of  the  yeasts,  in  the 
preparation  of  yeast  juice,  which  contains  zymase,  is  colored  black 
by  Grams  solution  and  safranin  while  the  juice  takes  a  red  tint  with 
the  same  reagents.  There  must  be  present,  then,  in  yeast  a  soluble 
albumin  which  may  be  colored  red  and  an  insoluble  albumin  which 
takes  a  black  color  according  to  this  procedure. 

There  has  been  proven  in  yeasts,  an  albuminoid  substance  soluble 
in  warm  alcohol,  which  must  be  a  peptone  produced  by  the  action  of 

1  Amato,  A.    The  lipoids  of  blastomycetes.     Cent.  Bakt.  Abt.  Ill,  42,  (1915) 
689-98. 

2  Bokorny,  Th.    Yeast  fat.     Allgem.  Brau-Hopfen  Ztg.  55,  (1915)  1803-5. 

3  Neville,  H.  A.  D.    The  fat  of  yeast.    Biochem.  Jour.  7,  341-348. 

4  Bokorny,  Th.    Accumulation  of  fat  in  plant  cells,  especially  in  yeasts.    Arch, 
physiol.  305-349,  1915;   Chemical  Abstracts,  II,  (1917)  260. 

6  Meisenheimer,  J.  Neue  Versuche  mit  Hefepressafs.  Zeit.  physiol.  Chemie, 
37,  1904. 


56  PHYSIOLOGY  OF  YEASTS 

endotryptase,  a  proteolytic  enzyme  which  we  shall  discuss  further 
on  in  this  book.  Also  derivatives  of  the  albuminoids  have  been 
demonstrated  such  as  the  amino  acids  (leucine  and  tyrosine);  these 
are  also  products  of  digestion  by  the  endotryptase. 

Schutzemberger l  has  demonstrated  the  purine  derivatives  (xanthine, 
hypoxan thine,  guanine).  Kossel2  and  Buchner  3  have  found  nucleic 
acids  in  rather  large  quantities. 

As  the  nucleus  of  yeasts  is  usually  small  and  poor  in  chromatin, 
Meyer  has  been  led  to  think  that  this  nucleic  acid  is  not  derived  entirely 
from  the  nucleus  but  also  from  the  protoplasm.  According  to  this  au- 
thor, the  metachromatic  corpuscles  which  are  so  abundant  in  yeasts 
result,  as  we  have  said,  from  a  combination  of  nucleic  acids  with  an  un- 
known organic  base.  Kohl  agrees  with  this  and  also  states  that  these 
bodies  represent  nucleo-proteins. 

Belohoubek  has  analyzed  yeasts  chemically  with  the  following  re- 
sults: 

Composition  Fresh  Yeast         Dry  Yeast 

Water 68.02                   

Nitrogenous  matter.  . 13. 10  40. 98 

Fatty  matter 0. 90                    2. 80 

Cellulose 1.75                    5.47 

Starchy  material 14. 10  44. 10 

Organic  matter 0. 34                    1 . 06 

Mineral  matter 1 . 77                    5. 54 

Miscellaneous 0. 02                   0. 05 

Jones4  has  given  some  attention  to  the  nucleic  acids  in  yeasts.  He 
prepared  the  potassium  salt  of  guanylic  acid  from  yeast.  In  another 
investigation,  two  dinucleotides  were  obtained,  one  yielding  guanine 
and  cytosine  and  the  other  adenine  and  uracil.  He  also  described  a 
compound  of  guanosine  and  guanylic  acid.  In  studying  the  struc- 
ture of  yeast  nucleic  acid,  Jones  and  Read5  hydrolyzed  yeast  nucleic 
acid  to  yield  a  dinucleotide  which  was  shown  to  be  adenine-uracil.  The 

1  Schutzemberger.     Les  Fermentations.     Paris,  1892. 

2  Kossel.    Ueber  das  Nuclein  der  Hefe,  Zeit.  physiol.  Chemie,  3. 

3  Buchner,  Ed.    Alkoholische  Garung  ohne  Hefezellen.     Berichte  der  deutsch. 
Gesellsch.  30,  1897. 

4  Jones,  W.     Formation  of  guanylic  acid  from  yeast  nucleic  acid.     J.  Biol. 
Chem.  12,  31-5.     The  partial  enzymic  hydrolysis  of  yeast  nucleic  acid.     J.  Biol. 
Chem.  17,  71-80.    Simpler  nucleotides  from  yeast  nucleic  acid,  J.  Biol.  Chem.  20, 
25-35  (1915). 

6  Jones,  W.,  and  Read,  B.  E.  Adenin-uracil  dinucleotide  and  the  structure 
of  yeast  nucleic  acid.  J.  Biol.  Chem.  29,  111-22.  The  mode  of  nucleotide  linkage 
in  yeast  nucleic  acid.  J.  Biol  Chem.  29,  123-6,  Uracil-cytosine  dinucleotide. 
J.  Biol.  Chem.  31,  39-45, 


^  GENERAL  PHENOMENA  OF  NUTRITION  57 

formation  of  such  a  salt  cannot  be  explained  if  the  two  mononucleotides 
are  joined  through  the  PO4.  Therefore,  the  nucleotides  in  this  dinu- 
cleotide,  and  those  in  nucleic  acid,  must  be  joined  through  the  carbohy- 
drate. This  was  also  verified  when  using  the  method  described  by  Jones 
and  Gehrmann ;  Levene  1  takes  exception  to  these  conclusions.  He  calls 
attention  to  the  fact  that  nucleotides,  forming  tetrabrucine  salts,  might 
be  linked  through  the  carbohydrate  group  of  one  and  through  the  base 
of  the  other.  He  emphasizes  that  further  work  is  necessary  before 
deciding  between  the  two  possibilities,  and  that  work  should  also  be 
carried  out  on  other  nucleic  acids  such  as  thymus  nucleic  acid.  Levene2 
in  continuing  this  work  secured  evidence  that  there  is  no  experimental 
proof  that  the  nucleotides  in  yeast  nucleic  acid  were  bound  together 
through  the  carbohydrate  group.  He  does  not  believe  that  a  tetra- 
riboseis  the  nucleus  of  yeast  nucleic  acid.  He  considers  that  the 
work  in  his  laboratory,  and  in  Jones',  indicates  the  tetranucleotide 
structure  of  nucleic  acid.  The  following  three  nucleotides  were  iso- 
lated in  pure  form:  guanylic  acid,  uredinephosphoric  acid  and  ade- 
nophosphoric  acid. 

Levene 3  in  later  investigations  has  written  the  structure  of  yeast 
nucleic  acid  as  follows : 

H0\ 

O  =  P  —  CgHiA.Cs^NsO 
HO/ 

H0\ 

O  =  P  —  C5H804.C4H4N30 
HO/ 

HO\ 

O  =  P  — 

HO/ 

H0\ 

0  =  P  — 
HO/ 


1  Levene,  P.  A.     The  structure  of  yeast  nucleic  acid.     J.  Biol.  Chem.  31, 
591-8,  1917. 

2  Levene,    P.     The   structure   of  yeast  nucleic   acid.     II.   Uridinephosphoric 
acid.    J.  Biol.  Chem.  33,  229-234. 

3  Levene,  P.  A.     The  structure  of  yeast  nucleic  acid.     Jour.  Biol.  Chem.  31 
(1917),  591-598;   The  structure  of  yeast  nucleic  acid.     II,  Uridinephosphoric  acid. 
Jour.  Biol.  Chem.  33  (1918),  229-234;  III,  Ammonia  hydrolysis.    J.  Biol.  Chem.  33 
(1918),  425-428. 


58  PHYSIOLOGY  OF  YEASTS 


General  Considerations  of  Enzymes  in  Yeasts 

The  study  of  enzymes  has  been  much  advanced  by  Buchner  who 
developed  a  procedure  which  allowed  the  extraction  of  the  juice.  Before 
this  method  was  known,  it  was  difficult  to  extract  these  enzymes  which, 
as  is  generally  known,  are  not  always  susceptible  to  passage  through  a 
membrane.  They  may  remain  on  the  interior  of  the  cell  and  act  there. 
Buchner's  method,  which  consists  in  searching  the  yeast  juice  for  the  en- 
zymes, is  the  only  one  which  offers  any  guarantee  of  success. 


Preparation  of  Yeast  Juice 

It  is,  then,  by  the  preparation  of  yeast  juice  that  we  must  take  up 
the  study  of  the  enzymes.  In  1897,  Buchner  was  able  to  isolate  the 
enzyme  which  produced  the  alcoholic  fementation  by  decomposing  sugar 
into  carbonic  acid  and  alcohol.  This  he  called  zymase  or  alcoholase.  He 
did  this  according  to  the  following  procedure.  He  ground  up  1000  grams 
of  yeast  after  careful  washing  and  drying.  This  was  a  difficult  proce- 
dure on  account  of  the  elasticity  of  the  yeast  cells,  but  in  order  to  ac- 
complish this,  it  was  necessary  to  mix  the  cells  with  fine  sand  and  rotten 
stone.  With  the  aid  of  a  heavy  iron  pestle,  he  triturated  the  yeast, 
previously  dehydrated,  with  1000  grams  of  quartz  sand  and  250  grams 
of  rotten  stone  in  the  form  of  a  thick  paste.  This  was  expressed  in  a 
hydraulic  press  under  a  pressure  of  300  to  500  atmospheres.  From  400 
to  500  c.c  of  the  yeast  juice  were  thus  obtained. 

The  extract  thus  obtained  is  a  brownish  liquid  with  somewhat  the 
odor  of  fresh  yeast  little  or  not  at  all  dialyzable.  Heating  to  40-50° 
causes  a  precipitation  of  albumin  and  the  liquid  loses  its  fermenting 
power.  It  contains,  along  with  a  certain  quantity  of  albumin  (4.15  per 
cent),  the  products  of  tryptic  digestion  (albumoses,  peptones,  tyrosine), 
lecithin,  a  phosphorus  compound  (nucleic  acid),  2  per  cent  of  ash  and 
the  products  of  fermentation  (0.53  per  cent  of  alcohol,  0.07  per  cent 
carbon  dioxide,  0.096  per  cent  of  glycerol,  and  0.016  per  cent  of  suc- 
cinic  acid). 

Along  with  the  albuminoids  precipitable  by  alcohol  and  coagulable 
by  heat,  are  various  enzymes  which  we  shall  take  up  further  on  (endo- 
tryptase,  maltase,  invertase,  glycogenase,  lipase,  etc.),  and  especially 
zymase;  but  this  has  not  been  isolated  in  the  pure  state.  When  placed 
with  fermentable  sugars  (saccharose,  maltose,  glucose,  levulose)  these 
sugars,  after  a  few  minutes,  are  changed  to  alcohol.  Further  on,  we 
shall  discuss  the  properties  of  this  enzyme. 


ENZYMES  OF  PROTEIN  SUBSTANCES  59 

ENZYMES   OF  PROTEIN   SUBSTANCES 
Proteases 

Geret  and  Hahn  have  shown  that  yeast  juice  dissolves  the  floccu- 
lent  coagulum  of  albuminous  materials  like  fibrin  or  coagulated  albumin. 
The  juice  contains  a  proteolytic  enzyme  or  endotryptase,  which  is  ca- 
pable of  being  isolated  in  comparatively  pure  state.  This  enzyme  plays 
a  very  important  role  in  the  life  of  the  cell.  The  investigations  of  Gro- 
mow  and  Gregoriew1  have  shown  that  this  endotryptase  exercises  a 
powerful  action  on  the  juice  itself  and  that  it  alters  and  digests  it  rapidly 
at  30-35°.  This  action  explains  the  rapidity  with  which  zymase  is 
destroyed.  This  enzyme  is  more  active  in  an  acid  than  in  an  alkaline 
medium,  being  favored  by  0.2  per  cent  of  hydrochloric  acid.  It  seems 
to  approach  trypsin  more  closely  in  characteristics  than  pepsin,  for 
Geret  and  Hahn  have  obtained  a  fairly  complete  degradation  of  albu- 
minoid substance  as  with  trypsin.  The  presence  of  leucine  and  tyrosine 
has  been  shown. 

Endotryptase  liquefies  gelatin  according  to  Hahn2  and  Hjort.  This 
is  an  intracellular  enzyme  and  not  able  to  pass  through  the  cell  mem- 
brane. This  fact  makes  it  difficult  to  understand  how  the  yeast  is  able 
to  liquefy  gelatin.  It  is  common  knowledge  that  the  yeasts  liquefy  and 
peptonize,  as  has  been  shown  by  the  works  of  Linder,  Boullinger,  Bei- 
jerinck  and  Astari.  Also  one  is  obliged  to  admit  with  Will  that  endo- 
tryptase, normally,  intracellular,  is  able  under  certain  conditions  to 
diffuse  through  a  membrane.  This  diffusion  occurs  at  various  phases 
of  its  development,  and  especially  in  cells  which  are  not  in  normal  con- 
dition (dead  or  diseased  cells). 

We  have  stated  that,  according  to  Boullinger,  certain  yeasts  se- 
crete a  casease.  The  formation  of  a  curd  in  milk  after  a  few  months 
was  determined.  This  coagulum  dissolves  little  by  little  and  the  liquid 
becomes  yellowish.  The  transformation  of  the  casein,  yields  tyrosine, 
leucine  and  other  ammoniacal  bodies.  Bochicchio  has  verified  the 
secretion  of  rennin  by  Lactomyces  inflans  caseigrana.  Rapp  has  ob- 
served the  presence  of  casease  in  certain  yeasts.  Dombrowski  has 
shown  that  many  of  the  yeasts  peptonize  milk  strongly,  especially 
S.  lactis  v. 

According  to  Boullinger,  there  is  a  relation  between   the  yeasts 

1  Gromow,  T.,  and  Gregoriew,  O.     Die  Arbeit  der  Zymase  und  der  Endo- 
tryptase in  den  abgetoteten  Hefezellen  unter  verschiedenen  Verhaltnissen.     Zeit. 
f.  physiol.  Chemie,  2,  1904. 

2  Hahn,  M.,  and  Geret,  Z.     Ueber  das  Hefe  Endotryptase,  Zeit.  Biol.  22, 
1900. 


60  PHYSIOLOGY  OF  YEASTS 

which  liquefy  gelatin  and  those  which  attack  casein.1  Those  which  de- 
stroy the  most  casein  liquefy  gelatin  most  rapidly.  Also  it  seems  very 
probable  that  the  liquefaction  of  gelatin  and  the  peptonization  of  casein 
are  due  to  the  action  of  endotryptase. 

According  to  Bokorny  and  Vines,  yeasts  contain  another  protease 
which  acts  like  pepsin;  but  the  existence  of  this  enzyme  is  rather 
obscure. 

Buchner  and  his  collaborators  have  isolated  from  yeast  juice  an 
enzyme  which  protects  albuminous  matter  from  the  action  of  endo- 
tryptase which  they  have  named  antiprotease.  This  enzyme  seems  to 
play  an  important  role  in  the  life  of  the  yeast;  it  governs  the  digestive 
functions  and  balances  the  action  of  the  proteases. 

Rennin :  The  investigations  of  Boullinger 2  have  indicated  the  pres- 
ence of  rennin  in  yeasts.  This  author  has  verified  the  existence  of  ren- 
nin  in  certain  yeasts  by  inoculating  skimmed  milk  and  after  a  few 
months  a  coagulum  formed  which  gave  evidence  of  the  presence  of 
rennin.  The  curd  eventually  dissociated  under  the  influence  of  casease. 

Bochicchio  has  stated  that  the  Lactomyces  inflans  caseigrana  pre- 
cipitated milk.  The  same  observation  has  been  reported  by  Dom- 
browski  for  other  milk  yeasts.  Other  yeasts  in  milk  do  not  cause  this 
change  (yeasts  of  Adametz,  Duclaux  and  Kayser).  On  the  other  hand, 
many  other  species  of  yeasts  (S.  glutinia  of  Sartory)  coagulate  casein 
without  digesting  it,  thus  secreting  rennin  but  not  casease.  (Valagussa 
and  Mafera.) 

Nucleases  or  Enzymes  of  Nucleo-proteins 

It  seems  also  that  yeasts  contain  enzymes  capable  of  decomposing 
nucleo-proteins  and  nucleic  bases.  Schutzemberger  has  shown  that 
xanthine,  hypoxanthine  and  guanine  are  found  among  the  autolytic 
products  of  yeasts.  Recently  Shiga,3  in  making  yeast  juice  act  on  a 
solution  of  guanine,  has  detected  a  decrease  in  the  quantity  of  this  base 
and  an  increase  in  the  xanthine  which  would  tend  to  prove  the  presence 
of  a  guanase.  According  to  the  same  author,  yeast  juice  may  also  con- 
tain arginase.  When  submitting  a  solution  of  arginine  to  the  action  of 
yeast  juice  in  the  presence  of  toluene,  Shiga  has  observed  a  disap- 

1  Diehl  (Jour.  Inf.  Dis.  24  (1919),  347-361)  has  reported  a  type  of  specificity 
among  the  bacterial  proteases.     This  author  was  able  to  detect  a  specificity  for 
proteins  with  certain  amino  acids,  after  the  bacterium  had  been  grown  on  media 
containing  these  amino  acids  as  the  only  source  of  organic  nitrogen. 

2  Boullinger,  E.    Action  de  la  levure  de  biere  sur  le  lait.    Ann.  Inst.  Pasteur, 
2,  1897. 

3  Shiga,  K.     Ueber  einige  emsige   Hafefermente,   Zeit.  physiol.  Chemie,   42, 
1904. 


CARBOHYDRATE   ENZYMES  61 

pearance  of  arginine  and  at  the  same  time  the  formation  of  ornithine 
or  urea.  But  guanine  is  not  decomposed  by  the  arginase  of  the  yeasts. 

Straughn  and  Jones  1  have  found  guanase  by  centrifuging  aqueous 
extracts  of  yeasts  made  according  to  the  following:  300  grams  of 
yeast  were  macerated  for  15  hours  in  a  liter  of  water  to  which  6  c.c  of 
chloroform  had  been  added.  The  liquid  thus  obtained  could  transform 
guanine  into  xanthine  and  therefore  contain  guanase;  it  could  not 
change  adenine  into  hypoxanthine  nor  hypoxanthine  into  xanthine. 
Consequently  it  did  not  contain  adenase  nor  xanthooxidase 

According  to  H.  Pringsheim  2  there  exists  in  yeasts  a  special  de- 
amidase,  which  permits  them  to  take  nitrogen  from  amino  acids 
without  the  production  of  ammonia  indicating  a  preliminary  decom- 
position. Finally,  Effront  has  recently  discovered  in  top  beer  yeasts 
an  amidase  which  acts  also  on  amino  acids  but  produces  ammonia 
and  volatile  acids. 

Lipase 

The  existence  of  a  lipase  in  the  cell  sap  which  transforms  fats 
into  fatty  acids  and  glycerol,  has  been  shown.  This  lipase  seems  to 
exert  an  injurious  action  on  the  zymase.  This  seems  to  be  composed 
of  a  proteolytic  enzyme,  properly  speaking,  and  of  a  coferment. 
Lipase  decomposes  the  coferment. 

Lipase  seems  to  be  intracellular  and  acts  on  the  fats  which  it  en- 
counters within  the  protoplasm  of  the  yeast,  especially  during  sporula- 
tion,  which  are  the  reserve  products  for  the  cell  during  maturation 
of  the  ascospores. 

In  certains  yeasts,  however,  lipase  is  able  to  diffuse  through 
membranes,  for  van  Tieghem  has  discovered,  some  time  ago,  S.  olei 
which  lives  in  oil  and  decomposes  it.  More  recently  Piedallu 3  has 
found  a  yeast  which  lives  in  oil  and  offers  the  same  properties.  Rogers 
and  Jensen 4  have  mentioned  very  many  Torula  which  decompose 
butter. 

Carbohydrate  Enzymes 

The  investigations  of  Fischer  and  Thierf elder  5  have  shown  that 
only  the  sugars  with  carbon  atoms  in  multiples  of  three  are  ferment- 

1  Straughn,  N.,  and  Jones,  W.     The  nuclein  ferments  of  yeasts.     Jour.  Biol. 
Chem.  6,  1909. 

2  Pringsheim,  H.     Ueber  Pilzdesamidase.     Biochem.  Zeitschr.  12,  1908. 

3  Piedallu,   A.     Sur  une  levure  qui  agit  sur  les  corps  gras.     Comp.  Rend. 
Soc.  de  Biol.  65,  1908. 

4  Jensen,  O.    Bakteriologische  Studien  iiber  danische  Butter.    Cent.  Bakt.  29, 
1911. 

5  Fischer,  E.,  and  Thierf  elder,  H.    Verhalten  der  verschiedenen  Zucker  gegen 
reine  Hefen.    Berichte  d.  deutsch.  Gesellschaft,  27,  1894. 


62      .  PHYSIOLOGY  OF  YEASTS 

able.  These  are  the  glyceroses  (C3H6O8),  tetroses  (C4H8O4),  hex- 
oses  (C6Hi206)  ,  nonoses  (C9Hi8O9)  ,  the  sugars  of  Ci2  and  ds  which  are 
bisaccharides  and  trisaccharides  and  finally  the  polysaccharides 
(starch,  inulin,  glycogen,  etc.).  It  is  known  that  the  bisaccharides  and 
trisaccharides  must  be  changed  to  hexoses  in  order  to  be  fermentable. 
Fermentation,  then,  consists  in  a  molecular  splitting,  in  the  course 
of  which  large  molecules  of  sugar  are  changed  into  molecules  which 
are  much  simpler,  the  bi-  or  trisaccharides  into  hexoses,  alcohol  and 
carbonic  acid. 

Polysaccharides:  Glycogenase,  Amylase,  Inulase:  According  to 
the  results  of  Wroblewsky,  Cremer,  Kohl  and  Hosaceus,  and  Geret 
and  Hahn  yeast  juice  contains  a  hydrolytic  enzyme  for  glycogen, 
glycogenase.  Yeasts  do  not  act  upon  glycogen  when  it  is  given  them 
as  food  because  the  glycogenase  is  an  intracellular  enzyme,  and 
glycogen  is  not  able  to  pass  through  the  cell  membrane.  This  glyco- 
genase is  able  to  act  only  upon  the  glycogen  which  is  made  by  the 
yeast  itself  on  the  interior  of  the  cell. 

Starch,  in  order  to  be  fermented,  must  be  transformed  into  dex- 
trine and  maltose;  then  the  dextrine  itself  is  changed  into  maltose. 
This  is  transformed  by  maltose  into  glucose  l  which  is  then  fermented. 
The  exact  mechanism  of  saccharification  is  not  known.  However, 
according  to  the  investigations  of  Maquenne  and  Roux  starch  is  com- 
posed of  from  90  to  92  per  cent  of  amylose  and  from  8  to  10  per  cent 
of  amylopectin.  The  change  of  starch  into  maltose  seems  to  demand 
the  action  of  three  enzymes,  amylase,  amylopectinase  and  dextrinase. 
The  amylase  changes  amylose  into  maltose,  the  amylopectinase 
changes  amylopectines  into  dextrines  and  dextrinase  changes  dex- 
trine to  fermentable  maltose. 

Some  yeasts  are  able  to  ferment  starch,  as  S.  exiguus  thermanti- 
tonum,  acetethylicus,  Sch.  Pombe,  mettacei,  octosporus,  the  yeast  of 
Logos  and  some  yeasts  of  Saaz  and  Mycoderma  sphaeromyces. 

Inulin  differs  from  starch  by  its  composition.  Certain  yeasts  are 
able  to  saccharify  it  and  ferment  it  on  account  of  an  inulase  which 
produces  levulose  and  not  maltose,  as  from  starch.  This  enzyme 
has  been  encountereed  in  Sch.  Pombe  and  mellacei,  S.  marxianus 
and  thermantitonum,  certain  species  of  the  type  of  Saaz,  and  the 
yeasts  E  and  F  of  Rose. 

Trisaccharides:  Raffinase,  Melibiase  and  Melizitase:  Raffinose 
or  melitriose  (Cis^Oie)  is  capable  of  decomposition  by  certain  yeasts. 


H20 

Raffinose  Levulose        Melibiose 

By  glucose  we  shall  mean  d-glucose  or  dextrose* 


CARBOHYDRATE   ENZYMES  63 

But  some  decompose  it  simply  into  levulose  and  melibioss,  causing 
only  the  levulose  to  ferment;  others  are  able  to  take  it  further.  They 
act  on  the  melibiose  which  they  change  to  dextrose  and  galactose; 
this  is  fermented  to  d-glucose.  The  dissociation  of  raffinose  is,  then, 
the  work  of  two  enzymes,  raffinase  and  melitriase,  which  split  the  raf- 
finose into  levulose  and  melibiose,  and  a  melibiase  which  splits  the 
melibiose  into  dextrose  and  galactose. 

Ci2H22Oii  +  H2O  =  C6H12O6  +  C6H12O6 
Melibiose  Dextrose       Galactose 

Saccharomycodes  Ludwigii,  S.  marxianus,  exiguus,  thermantitonum, 
cartilaginosus,  Sch.  Pombe,  mellacei,  octosporus,  the  yeasts  E  and  F  of 
Rose,  and  the  yeast  of  Logos  cause  raffinose  and  levulose  to  ferment 
but  not  melibiose.  They  contain  only  a  raffinase.  On  the  contrary, 
bottom  yeast  of  the  Frohberg  and  Saaz  types  cause  melibiose  and 
levulose  to  ferment  while  the  top  yeasts  of  these  types  are  able  to 
ferment  .only  levulose  and  do  not  possess  a  melibiase. 

According  to  Kalanthar,  a  melizitase  exists  in  some  yeasts  which 
decomposes  melizitose  into  dextrose  and  turanose. 


+  H2O  =  CeH^Oe  +  Ci2H22On 
Melizitose  Glucose        Turanose 

Disaccharides  :  Sucrase,  Maltase,  Lactase,  Trehalase:  The  di- 
saccharides  possess  the  general  formula  Ci2H22On.  Four  of  them, 
saccharose,  maltose,  lactose  and  trehalose,  are  well  known. 

Saccharose  is  changed  by  sucrase  or  invertase  to  glucose  and  levu- 
lose. The  phenomenon  may  be  expressed  by  the  following  equation: 

Ci2H220n  +  H2O  =  CeH^Oe  +  CeH^Oe 
Saccharose  Glucose        Levulose 

It  was  in  the  yeasts  that  Berthelot  found  sucrase  for  the  first  time. 
It  is  rather  widely  distributed  among  them.  In  certain  species,  this 
enzyme  remains  inside  of  the  cell  and  only  that  sucrose  which  passes 
into  the  cell,  is  decomposed.  The  glucose  and  levulose  thus  formed 
diffuse  through  the  membrane  into  the  medium.  But  in  Monilia 
Candida  and  in  the  yeast  of  "Soja"  not  only  the  sucrase  remains  in 
the  cell  but  also  the  glucose  and  levulose,  whether  it  is  not  able  to 
diffuse  or  whether  it  is  destroyed  as  soon  as  it  is  formed.  Thus  it 
is  impossible  to  observe  the  inversion  of  sucrose  by  an  analysis  of  the 
fermentation  mixture.  But  in  many  yeasts,  sucrase  is  diffusible,  and 
is  able  to  be  secreted  outside  of  the  cell.  Finally  certain  yeasts,  as 
Sch.  octosporus,  S.  apiculatus,  Behrensianus,  Rouxii,  mali  Duclauxi, 
P.  membrancefaciens,  W.  belgica,  do  not  possess  sucrase  and  are,  conse- 
quently, unable  to  ferment  sucrose. 


64  PHYSIOLOGY  OF  YEASTS 

Maltose  in  order  to  be  assimilated  and  fermented  must  also  be 
decomposed  by  an  enzyme  into  two  molecules  of  glucose.  This  is 
accomplished  by  maltase. 

C12H220U  +  H20  =  C6H1206  +  C6H1206 

Maltose  Glucose         Glucose 

Many  yeasts  at  once  decompose  maltose  and  saccharose  (S.  cere- 
visiae  Pastorianus,  intermedius,  validus,  ellipsoideus,  and  turbidans). 
On  the  contrary,  S.  marxianus,  exiguus,  Jorgensenii,  Saccharomy- 
codes  Ludwigii  and  Saccharomyces  guttulatus,  are  able  to  ferment  sac- 
charose but  do  not  ferment  maltose.  Maltase  is  then  a  different 
enzyme  from  sucrase.  Other  yeasts  such  as  S.  apiculatus  ferment 
neither  maltose  nor  saccharose,  and  thus  possess  neither  maltase  nor 
sucrase.  Maltase  is  a  reversible  enzyme  and  transforms  maltose 
into  isomaltose. 

For  lactose  to  undergo  alcoholic  fermentation,  it  must  first  be 
changed  by  lactase  into  glucose  and  galactose. 

Ci2H22On  +  H20  =  CeHiijOe  + 


Yeasts  possessing  a  lactase  are  not  common.  Only  a  small  number 
are  known.  Lactase  has  been  found  in  S.  Kephir  (Beijerinck),  tyrocola 
fragilis,  acidi  lactici,  lactis  a  and  ft  (Dombrowski),  Zyg.  lactis,  the 
yeasts  of  Duclaux,  Adametz,  and  Kayser,  and  various  Torula  and 
Mycoderma  isolated  by  Dombrowski,  etc.  Hunter  l  has  mentioned  a 
yeast  which  was  able  to  ferment  the  lactose  in  cream.  This  yeast 
apparently  possessed  a  lactase.  Hunter  also  reviews  the  literature 
on  yeasts  which  possess  this  enzyme.  Several  such  instances  are  men- 
tioned. 

Trehalose  is  decomposed  by  trehalase  into  glucose  and  levulose. 
Many  yeasts  seem  to  possess  a  trehalase  and  are  thus  able  to  hydrolyze 
trehalose. 

Kalanthar  has  found  it  in  many  beer  and  wine  yeasts.  S.  ther- 
mantitonum  and  the  bottom  yeast  of  Frohberg  (Linder)  also  contain 
trehalase. 

Neuberg  and  Karczag2  found  that  pyroracemic  and  oxymalic  acids 
were  fermented  with  the  formation  of  carbon  dioxide.  Acet  alde- 
hyde was  identified  as  the  other  product.  This  would  indicate  that 
a  carboxylase  removed  the  CO2  from  the  pyroracemic  acid.  Carbon 
dioxide  was  also  split  from  the  following  acids:  acetone  dicarboxylic, 
chelidonic,  dihydroxytartaric,  phenylglyoxylic  and  acetylenedicar- 

1  Hunter,  O.  W.      A  Lactose  Fermenting  Yeast  Producing  Foamy   Cream. 
Journal  of  Bacteriology,  3  (1918)  293-300. 

2  Neuberg,  C.  and  Karczag,  L.    Carboxylase,  a  new  enzyme  of  yeast.     Bio- 
chem.  Zeit.  36,  68-75.  76-81;   Chem.  Abstracts,  6  (1912),  380. 


CARBOHYDRATE   ENZYMES  65 

boxy  lie  acid.  Neuberg  and  Czapski *  demonstrated  the  presence  of 
carboxylase  in  the  juice  of  top  yeast.  Bau 2  studied  the  action  of 
yeast  on  pyroracemic  acid  in  the  presence  of  certain  inorganic  salts. 
No  carboxylase  could  be  found,  which  confirmed  Neuberg's  theory 
that  this  enzyme  does  not  diffuse  from  living  yeast  into  the  surround- 
ing medium.  Later  Bau  3  stated  that  carboxylase  could  be  demon- 
strated in  dried  yeast  20  years  old.  Other  enzymes  such  as  invertase, 
maltase,  melibiase,  emulsin,  amygdalase,  lipase  and  oxidase  were 
found. 

Glucosides:  Emulsin:  Fischer  and  Thierfelder  have  shown  that 
some  yeasts  are  able  to  split  the  a-methylglucosides  (substances  ob- 
tained from  a  condensation  of  methyl  alcohol  with  glucose)  into 
methyl  alcohol  and  glucose.  They  are  not  able  to  split  the  /3-methyl- 
glucosides,  however. 

According  to  Fischer,  this  action  is  not  brought  about  by  a 
special  enzyme,  but  by  maltase  which  possesses  the  ability  of  splitting 
both  the  a-methylglucosides  and  maltose.  On  the  contrary,  the 
/3-methylglucosides  are  not  decomposed  except  by  an  emulsin  which 
acts  to  break  up  this  substance.  The  investigations  of  Bresson 4 
seem  to  prove,  on  the  contrary,  the  existence  of  a  special  enzyme  in 
the  yeasts  of  Frohberg,  which  is  sharply  set  apart  from  sucrase  and 
maltase,  and  a-methylglucase. 

Whatever  is  the  truth,  many  yeasts  are  known  which  are  able  to 
ferment  the  a-methylglucosides.  Some  of  these  are  Sch.  octosporus, 
Pombe,  mellacei,  S.  thermantitonum,  the  yeast  of  Logos,  the  yeast 
of  Frohberg,  and  certain  yeasts  of  Saaz. 

The  investigations  of  Henry  and  Auld  5  have  indicated  that  when 
yeast  acts  on  amygdaline  in  the  presence  of  toluene  at  40°,  after  5 
days  about  33  per  cent  of  the  glucoside  is  decomposed  and  after  11 
days,  67  to  70  per  cent.  From  this,  these  authors  are  led  to  believe 
that  an  emulsin  exists  in  yeasts.  Now  it  seems  that  certain  yeasts 
may  act  upon  the  /3-methylglucosides  which,  according  to  Fischer, 
are  not  decomposed  by  emulsin. 

1  Neuberg,  C.  and  Czapski,  L.     Carboxylase  in  the  juice  of  top  yeast.     Bio- 
chem.    Zeit.  67,  9-11,  1914.    Chem.  Abstracts,  9  (1915),  472. 

2  Bau,   A.     Carboxylase.     Wochenschr.   Brau.   32,   405-6.     Chem.  Abstracts, 
11  (1916),  797. 

3  Bau,  A.    Yeast  carboxylase;   its  permanence  in  a  dry  state  as  compared  with 
the  other  enzymes  of  yeast.     Biochem.  Zeit.  73,  340-368. 

4  Bresson.    Sur  Texistence  d'une  methylglucocase  specific  dans  la  levure  de 
biere.     Comp.  Rend.  Acad.  Sci.  151,  1910. 

6  Henry,  A.,  and  Auld,  M.  On  the  probable  existence  of  emulsin  in  yeast. 
Proc.  Roy.  Soc.  76,  1905. 


66  PHYSIOLOGY   OF  YEASTS 

Bau  1  has  shown  that  amygdalase  and  emulsin  are  present  in  Froh- 
berg  yeast.  Experiments  with  Saaz  yeast  indicated  amygdalase,  but 
no  emulsin.  S.  Ludwigii,  a  yeast  with  no  maltase,  acted  toward 
amygdalin  as  did  Frohberg  yeast.  This  seems  to  indicate  that  the 
disaccharide  complex  of  amygdalin  is  not  identical  with  maltose  though 
it  contains  two  dextrose  residues.  Bokorny  2  determined  the  presence 
of  amygdalin  in  brewers'  yeast  by  the  odor  of  oil  of  bitter  almonds 
in  incubating  a  mixture  of  yeast  and  amygdalin.  The  existence  of 
a  yeast  myrosinase  was  also  indicated  by  yeast  and  myrosin.  The 
glucosides,  arbutin,  ciniferin,  and  salicin,  were  not  changed  by  the 
yeast.  Farber 3  stated  that  amygdalase,  prunase  and  oxynitrilase, 
the  three  enzymes  necessary  for  the  complete  hydrolysis  of  amyg- 
dalin, could  be  separated  from  bottom  yeast. 

Oxidizing  and  Reducing  Enzymes 

Catalases  are  enzymes  which  decompose  hydrogen  peroxide  with 
the  formation  of  inactive  molecular  oxygen.  They  seem  to  play  a 

2  H2O2  =  H20  +  02 

role  in  regulating  the  production  of  hydrogen  peroxide  and  prevent- 
ing an  accumulation  of  it.  Buchner  was  the  first  to  detect  catalase 
in  yeast  juice. 

The  investigations  of  Tolomei,  Issajew,4  Low  5  Henneberg,  Neu- 
mann and  Wender,  have  confirmed  the  existence  of  this  enzyme. 
According  to  Neumann  and  Wender,  two  catalases  exist  in  yeast, 
an  a-catalase  insoluble  (?)  in  water  and  a  /3-catalase  soluble  in  water. 
These  enzymes,  which  are  found  in  yeast  juice  and  in  yeasts  killed  by 
antiseptics,  decomposed  hydrogen  peroxide  with  the  formation  of  free 
oxygen. 

Another  enzyme  similar  to  catalase,  pkilothion,  has  been  pointed 
out  by  Rey-Pailhade.  It  decolorized  methylene  blue  and  indigo  car- 
min  and  transformed  the  sulfur  in  hydrogen  sulfide,  and  the  icdin 
in  hydriotic  acid.  Grliss6  has  observed  this  same  enzyme  and  called 
it  hydrogenase. 

1  Bau,  A.      Behavior  of  amygdalin   towards   fermentation  organisms.      Wo- 
chenschr.  Brau.  34,  29-31  (1917);   Chem.  Absts.  12,  403  (1918). 

2  Bokorny,  T.     Emulsin  and  myosin  in  the  compressed  yeast  from  Munich 
brewery,  partly  also  in  baker's  yeast.    Biochem.  Zeit.  75,  376-416. 

3  Farber,  E.     Occurrence  of  emulsin-like  enzymes  separable  from  yeast  cells 
in  bottom  yeast;    also,  the  absence  of  myrosine  in  Berlin  top  and  bottom  yeast. 
Biochem.  Zeit.  78,  264-72.    Chem.  Absts.  11,  1658  (1917). 

4  Issajew,  W.     Ueber  Hefen  Katalase.      Zeit.  physiol.  Chemie,  44,  1905. 

6  Low,  O.    Zur  Unterscheidung  zwei  Arten  Katalase.     Cent.  Bakt.  10,  1903. 
6  Griiss,  J.     Ueber  Oxydaseerscheinungen  der  Hefe.     Woch.  fur  Brauerei,  17, 
1903. 


TOXINS  67 

Hydrogenase  seems  to  be  rather  widespread  among  the  yeasts. 
One  hundred  and  forty  years  ago,  Nessler  stated  that  if  flowers  of 
sulfur  were  added  to  a  liquid  undergoing  alcoholic  fermentation,  hy- 
drogen sulfid  would  be  formed.  We  shall  see  further  on  that  accord- 
ing to  Griiss,  hydrogenase  plays  a  role  in  alcoholic  fermentations. 

Reductases  for  other  sulfur  compounds  have  been  studied  by  vari- 
ous investigators.  Beijerinck l  and  Kossowicz  and  Loew  2  were  un- 
able to  find  any  reduction  of  sulfates  with  different  strains  of  yeasts. 
Among  the  strains  which  were  used  by  these  investigators,  were  Sac- 
charomyces  cerevisiae  and  Saccharomyces  ellipsoideus.  Tanner,3  how- 
ever, demonstrated  sulfate  reduction  with  9  out  of  30  pure  cultures 
of  yeasts.  The  fungi,  used  by  Tanner,  could  also  split  hydrogen 
sulfur  from  other  sulfur  compounds.  Most  of  the  cultures  could 
attack  the  sulfur  in  sodium  thiosulfate  and  a  few  reduced  the  sulfur 
in  sodium  sulfite.  Free  sulfur  was  also  changed  to  hydrogen  sulfide. 

Oxydases  are  enzymes  which  oxidize  and  yield  peroxides.  Grtiss 
has  pointed  out  the  presence  in  yeast  of  an  oxidase  which  does  not 
act  on  guaiac  but  gives  a  violet  reaction  with  tetramethylphenylen- 
diamine.  This  enzyme  oxidized  aldehydes  to  acetic  acid  and  reduced 
fuchsin  and  methylene  blue. 

It  is  undoubtedly  due  to  this  enzyme  that  certain  yeasts  are  able 
to  oxidize  alcohol  in  contact  with  air.  Grtiss  believes  that  they  play 
a  large  role  in  respiration. 

Toxins 

Haydruck  was  the  first  to  point  out  the  existence  in  yeasts  of  an 
endotoxin  capable  of  killing  them  when  it  is  extracted  from  the  cells 
and  introduced  into  the  culture  media.  Fernbach  4  and  Vulquin  6  have 
confirmed  the  existence  of  this  toxin  which  seems  to  play  toward  the 
yeast  the  role  of  an  antiseptic.  These  authors  have  prepared  this 
substance  in  the  following  manner:  Compressed  yeast,  previously 
dried  at  70°,  is  macerated  in  a  1  per  cent  solution  of  hydrochloric 
acid  for  about  20  hours  at  35-37°.  The  filtered  macerated  mixture 
is  evaporated  under  reduced  pressure,  having  been  slightly  alka- 

1  Beijerinck,  M.  W.     Cent.  Bakt.  Parasitenk.  Abt.  II,  6,  194-206,  1900. 

2  Kossowicz  and  Loew.     Garungsphysiol.  2,  87-103  (1912). 

3  Tanner,  F.  W.     Studies  on  the  Bacterial  Metabolism  of  Sulfur.      II.  For- 
mation of  hydrogen  sulfid  from  certain  sulfur  compounds  by  yeast-like  fungi.     Jour. 
Amer.  Chem.  Soc.  60  (1918),  663-9. 

4  Fernbach,  A.     Sur  un  poison  elabore  par  la  levure.     Comp.  Rend.  Acad. 
Sci.  144,  1909. 

6  Fernbach,  A.,  and  Vulquin,  E.  Quelques  observations  nouvelles  sur  le 
pouvoir  bacte"ricide  des  macerations  de  levures.  Comp.  Rend.  Acad.  Sci.  67,  1909. 


68  PHYSIOLOGY  OF  YEASTS 

linized  with  sodium  hydroxids.  The  distillate  is  received  in  dilute 
sulfuric  acid,  and  there  results  a  liquid  which,  after  neutralization,  pos- 
sesses toxic  properties  for  the  cells  when  introduced  into  them.  Ex- 
periments with  B.  coli  and  Staphylococcus  pyogenes  aureus  indicate  that 
the  yeast  toxin  is  also  poisonous  to  them.  Like  other  toxins,  it  passes 
through  porcelain  filters  and  is  destroyed  at  100°.  It  is  also  vola- 
tile. From  some  of  its  characteristics,  it  seems  that  this  toxin  ought 
to  belong  to  the  amines.  Fernbach  1  continued  his  study  of  toxic  sub- 
stances in  yeasts  by  drying  yeast  cells  at  37°  C.  and  extracting  them 
with  dilute  hydrochloric  acid.  The  filtered  extract  was  toxic  to 
yeasts  and  bacteria.  The  toxic  substance  was  destroyed  at  10°  C. 
and  was  volatile.  Haydruck 2  has  been  unable  to  confirm  these 
results  of  Fernbach.3 

NUTRITION  OF  YEASTS 
1.  Mineral  Elements 

Mayer 4  has  studied  the  mineral  elements  which  are  necessary  for 
the  yeasts.  This  author  attempted  to  determine  new  media  by  the 
introduction  of  new  elements.  The  mineral  mixture  which  yields  the 
bests  results,  is  as  follows: 

0.1    gram  Monobasic  potassium  phosphate 
0.1       "       Magnesium  sulfate 
0.1       "       Tribasic  calcium  phosphate 
100.00  c.c       Distilled  water 
15.00  grams  Candied  sugar. 

According  to  these  investigations,  potassium  phosphate  plays  an 
important  role,  after  which  is  magnesium.  It  is  interesting  to  note 
that  these  mineral  elements  are  the  same,  and  almost  in  the  same 
proportion,  as  those  which  have  been  found  by  analysis  of  yeasts. 
The  synthetic  method  has  then  confirmed  the  results  of  the  analytic 
method. 

The  investigations  of  Elion  5  and  Stern  6  have  confirmed  the  re- 
sults of  Mayer  and  shown  that  the  phosphates,  magnesium,  potas- 
sium and  sulfur  are  indispensable  elements  in  the  life  of  yeasts. 

1  Fernbach,  A.  The  poison  elaborated  from  yeast.  Comp.  Rend.  Acad.  Sci. 
149,  437-439. 

Haydruck,  F.    Yeast  poison  in  yeast.    Wochenschr.  Brau.  26,  677. 

Fernbach,  A.  Toxic  substance  elaborated  by  yeast.  Ann.  Brasserie  dis- 
tille  ie,  12,  361.  Chem.  Absts.  4  (1910),  948. 

Mayer,  A.     Lehrbuch  der  Garungschemie,  1902,  5th  edition. 

Elion,  H.    Studien  uber  Hefe.    Cent.  Bakt.  14,  1893. 

Stern.    Nutrition  de  la  levure.    Jour.  Chem.  Society,  1899  and  1901. 


NUTRITION  OF  YEASTS  69 


2.  Nitrogenous  Substances 

The  nitrogenous  substances  may  be  divided  into  four  groups: 
ammonia,  nitrates,  albumins,  and  their  derivatives,  such  as  amides 
and  amines.  Since  the  investigations  of  Boussingault,  it  has  been 
known  that  the  nitrates  play  an  important  role  in  the  nutrition  of 
higher  plants.  The  investigations  of  Muntz  have  shown  that  ammonia 
is  also  assimilated  by  higher  plants,  but  it  is  only  a  substance  of 
medium  importance.  In  the  nutrition  of  yeasts,  ammonia  salts 
(phosphates  and  sulfates),  on  the  contrary,  play  an  important  role  while 
the  nitrates  are  generally  not  assimilated. 

Pasteur  was  the  first  to  establish  that  the  ammonium  salts  were 
good  foods  for  the  yeasts.  The  later  investigations  by  Duclaux  and 
Laborde,  and  Laurent  have  confirmed  these  results.  The  experi- 
ments of  Laurent  have  indicated  that  yeasts  do  not  assimilate  ni- 
trates. According  to  this  author,  they  would  have  to  reduce  the 
nitrates  to  nitrites,  substances  which  are  toxic.  Beijerinck,  however, 
has  stated  that  certain  yeasts,  such  as  S.  acetethylicus,  are  able  to 
assimilate  nitrates.  Since  then,  the  investigations  of  Kayser,1  and 
Fernbach  and  Lanzenberg2  have  shown  that,  if  nitrates  are  injuri- 
ous to  multiplication,  they  have  a  favoring  influence  on  the  zymase 
in  fermentation. 

The  relation  of  albuminoid  substances  to  the  metabolism  of 
yeasts  is  very  obscure.  According  to  Pasteur  and  Ad.  Mayer,  the 
yeasts  are  unable  to  use  egg  white  or  blood  fibrin.  These  substances 
do  not  pass  through  the  cell  membrane,  and  the  endotryptase  of  the 
yeasts  is  an  intracellular  enzyme  which  does  not  pass  easily  to  the 
outside  of  the  cell.  We  have  seen  that,  according  to  Boullinger,  cer- 
tain yeasts  inoculated  into  milk  develop  very  slowly  and  produce, 
after  a  few  months,  a  curd  which  slowly  liquefies  with  the  formation 
of  ammonium  salts,  tyrosine,  and  leucine.  There  is  then  a  dissolu- 
tion and  digestion  of  the  casein  by  the  yeast.  It  is  known,  on  the  other 
hand,  that  certain  species  of  yeasts  liquefy  gelatin.  It  must  be  ad- 
mitted that,  under  special  conditions,  endotryptase  may  pass  through 
the  cell  membrane. 

On  the  contrary,  if  yeasts  do  not  accommodate  themselves  to 
these  compounds,  they  easily  assimilate  the  dialyzable  derivatives  of 
them,  such  as  the  albumoses  and  peptones.  It  is  curious  to  note  that 

1  Kayser,  E.     Influence   des   nitrates   sur   les   ferments   alcooliques.     Comp. 
Rend.  Acad.  Sci.  150,  1910. 

2  Fernbach,  A.,  and  Lanzenberg,  A.     De  1'Action  des  nitrates  dans  la  fermen- 
tation alcoolique.     Comp.  Rend.  Acad.  Sci.  151,  1910. 


70  PHYSIOLOGY  OF  YEASTS 

they  are  able  to  utilize  also  as  sources  of  nitrogen,  certain  enzymes 
such  as  pepsin  (Mayer  and  Heinzelmann) . 

According  to  more  recent  investigations,  the  derivatives  of  al- 
buminoids (amides,  amino  acids,  and  leucomaines)  are  assimilated 
more  easily  than  the  albumoses  and  make  up  the  desirable  nitrog- 
enous substances  for  the  yeasts.  The  work  of  Rettger  has  shown  the 
same  thing  for  the  bacteria. 

Waterman  l  stated  that  the  amino  group  is  an  especially  suitable 
source  of  nitrogen.  This  depends  on  the  presence  of  one  or  more 
acid  amide  groups  which  are  not  available  for  nutrition.  Waterman 
points  out  that  this  selective  action  of  yeasts  may  be  used  to  separate 
closely  related  compounds.  Asparagin  and  aspartic  acid  are  utilized 
while  succinamic  and  succinamide,  which  contain  only  the  acid  amide 
groups,  are  not  assimilated.  Cinnamamide  is  not  assimilated  while 
a-aminocinnamamide  is  used. 

Neubauer  and  Fromherz2  fermented  dl  phenylaminoacetic  acid 
(C6H4OH(NH2)COOH)  in  the  presence  of  10  per  cent  of  cane  sugar 
for  three  days.  There  was  left  an  amount  of  undecomposed  acid 
which  was  usually  more  or  less  1.  By  means  of  certain  methods  phenyl- 
glyoxylic  acid  hydrazone  was  obtained.  Sodium  succinate,  benzyl 
chloride,  p-hydroxyphenyl  ethyl  alcohol  (perhaps  from  yeast  tyro- 
sine),  /-acetylphenylamino  acetic  acid  were  obtained.  Para-hydroxy- 
phenylpyruvic  acid  was  also  fermented.  These  authors  are  led  to 
construct  the  path  from  amino  acid  to  the  next  lower  alcohol  as  fol- 
lows: 

RCH(NH2)COOH  =  RC(OH)(NH2)COOH 

=  RCOCOOH  =  RCHO=RCH2OEL 

The  processes  thus  involved  are  oxidation,  decarboxylation,  acid 
reduction.  The  alcohol  acid  RCHOHCOOH  and  the  acetylamino 
acid  result  from  secondary  reactions. 

Kossowicz 3  found  that  yeasts  could  utilize  nitrates.  Bokorny 4 
found  that  nitrates  were  unaltered  and  not  assimilated.  The  simple 
amines,  such  as  ethyl  amine,  were  also  unfavorable.  The  presence 
of  sugars  was  found  to  be  necessary  to  keep  down  the  bacteria.  With 
various  sources  of  nitrogen  in  the  medium,  the  following  increases 
were  observed  in  dried  yeast: 

1  Waterman,  H.  J.    Nitrogen  nutrition  of  compressed  yeast.    Zent.  Biochem, 
Biophys.  16,  276. 

2  Neubauer,  O.,  and  Fromhers,   K.     The  decomposition  of  amino   acids  in 
yeast  fermentation.    Zeit.  physiol.  Chem.  70,  326-350. 

3  Kossowicz,  A.     Behavior  of  yeasts  and  molds  towards  nitrates.     Biochem. 
Z.  67,  400-19,  1914. 

4  Bokorny,   Th.     Sources  of    nitrogen    of    yeasts.     Chem.   Ztg.   40    (1916), 
366-368. 


NUTRITION  OF  YEASTS  71 

(NH4)2  +  sucrose  =    71.8  per  cent  increase 

+  dextrose  =  113.0  "  " 

Asparagin  +  sucrose  =  103.7  "  "          " 

Aspartic  acid  +       "  61.3  "  " 

Leucine+       "  =    90.3  "  " 

Tyrosine+       "  =    61.3  "  " 

Glycine  +       "  =    25.8  "  " 

With  somatose  (flesh  albumose)  there  was  a  decrease  of  9.7  per 
cent  which  would  seem  to  indicate  that  the  albumoses  must  be  fur- 
ther decomposed.  Peptone  with  sucrose  gave  an  increase  of  177  per 
cent  while  peptone  alone  gave  152  per  cent. 

Lindner  and  Wtist x  find  that  urea  can  serve  yeasts  and  molds  as 
sources  of  nitrogen  but  not  for  carbon.  Bokorny2  has  measured  the 
increase  in  development  of  yeast  when  nitrogen  is  taken  from  urea, 
by  the  dry  weight.  Considerable  growth  took  place  when  the  yeast 
was  grown  in  urine  to  which  sugar  had  been  added.  The  urea  and 
not  the  hippuric  acid  is  said  to  be  the  source  of  the  nitrogen. 

Hoffman  3  has  shown  that  the  addition  of  ammonium  chloride  to 
bread  dough  saved  30  per  cent  of  the  yeast  ordinarily  used.  Experi- 
ments were  carried  out  which  showed  that  this  nitrogen  went  to  con- 
struct yeast  protein.  Good  arguments  are  presented  which  show 
that  this  salt  does  not  go  for  other  purposes.  Ehrlich  4  stated  that 
ammonium  salts  were  readily  changed  into  yeast  protein.  Delbrtick 
and  Classen  5  have  used  ammonium  salts  for  cultivating  yeast.  Voltz 6 
found  that  the  composition  of  yeast  grown  on  mineral  salts  was  like 
that  grown  with  other  sources  of  nitrogen. 

Kossowicz  and  Groller 7  have  stated  that  the  thiocyanates  will 
serve  yeasts  as  source  of  nitrogen  and  sulfur  but  not  carbon. 

1  Lindner,    P.,  and  Wust,  G.      Assimilation   of   urea  by   yeasts  and   molds. 
Wochenschr.  Brau.  30,  477-9.     Chem.  Absts.  8  (1914),  727. 

2  Bokorny,  Th.     The  increase  in  dry  weight  of  yeast  when  urea  is  used  as 
the  source  of  nitrogen.     Biochem.  Zeit.  82  (1917),  359-390;  The  culture  of  yeast 
in  the  presence  of  air  with  the  use  of  urea  as  source  of  nitrogen  and  with  different 
sources  of  carbon.     The  quotient  of  sugar  assimilation.     Biochem.  Z.  83  (1917), 
133-164.     Chem.  Absts.  12  (1918),  1203. 

3  Hoffman,  C.  H.     The  utilization  of  ammonium  chloride  by  yeast.     Jour. 
Ind.  Eng.  Chem.  9  (1917)  148-151. 

4  Ehrlich.     Biochem.  Z.  18,  391-423. 

6  Delbriick.  Classen.  A  new  method  for  increasing  the  production  of  yeast. 
Z.  Ver.  Deut.  Ing.  59  (1915),  844. 

6  Voltz.     Utilization  of  the  animal  organism  of  yeast  produced  from  sucrose 
and  nutritive  mineral  salts.    Z.  Spiritusind.  38  (1915)  235-6. 

7  Kossowicz,  A.  and  Groller,  L.    Thiocyanates  as  a  source  of  carbon,  nitrogen 
and  sulfur  for  molds,  yeasts  and  bacteria.     Zeit.  Garungsphysiologie,   2,  59-65. 
Chem.  Absts.  7  (1913),  808. 


72  PHYSIOLOGY  OF  YEASTS 

The  investigations  of  Mayer,  Haydruck  and  Kusserow,  Schulz, 
Thomas  1  and  Lindner,  have  shown  that,  among  the  amides,  allan- 
toin,  asparagin,  and  urea  are  assimilable  by  yeasts.  It  has  been 
pointed  out,  however,  that  the  investigations  of  Shiga,  Straughn  and 
Jones  have  indicated  in  yeast  juice  the  presence  of  a  guanase  which 
transforms  guanine  into  xanthine,  and  of  an  arginase  which  will  split 
xanthine  and  ornithine  into  urea.  According  to  Mayer,  caffein, 
creatin,  and  creatinin  are  not  acted  upon  by  yeasts. 

The  investigations  of  P.  Lindner,2  and  his  collaborators  Rlilke  and 
Hoffmann,  have  shown  that  yeasts  may  assimilate  products  of  their 
autodigestion.  Among  those,  tyrosine,  leucine,  adenine,  asparagine, 
aspartic  acid  and  ammonium  sulfate  are  most  easily  assimilated; 
finally,  choline  is  used.  All  of  the  yeasts,  however,  do  not  act  in  the 
same  manner  in  this  relation.  It  is  thus  that  the  top  and  bottom  yeast 
of  the  brewery  and  distillery  types  and  yeast  juice  easily  assimilate 
tyrosine,  leucine,  adenine,  aspartic  acid,  guanidine,  arginine,  hypo- 
xanthine,  histidine,  uracil,  choline,  thymine,  potassium  nitrate  and 
ammonium  sulfate.  Mycoderma  and  the  species  of  the  genus  Willia 
and  Pichia  use  almost  all  of  the  products  of  autolysis. 

More  recently,  Ehrlich  3  has  stated  that  leucine  and  isoleucine 
are  especially  desirable  compounds  for  yeasts  which  add  a  molecule 
of  water,  splitting  them  into  isoamyl  alcohol  (or  amyl)  and  ammonia. 
The  ammonia  is  used  by  the  yeast.  Effront 4  has  also  given  evidence 
of  the  existence  of  an  amidase  which  acts  on  amino  acids  but  without 
giving  alcohols,  transforming  them  into  ammonia  and  volatile  acids. 
Pringsheim 5  admits  the  existence  of  a  desamidase  which  allows  yeasts 
to  take  their  nitrogen  from  amino  acids  but  without  the  production 
of  ammonia.  The  investigations  up  to  the  present,  then,  seem  to  in- 
dicate that  the  amino  acids  make  up  a  better  source  of  nitrogen  for 
yeasts.  Data  from  Rettger's  laboratory  allow  similar  conclusions  for 
the  bacteria.  Koser  and  Rettger  6  have  shown  that  the  amino  acids 

1  Thomas,  P.    Sur  la  nutrition  azotee  de  la  levure.     Comp.  Rend.  Acad.  Sci. 
131,  1910. 

2  Lindner,  P.    Riilke,  P.,  and  Hoffmann.      Wochenschr.  Brauerei,  22,  No.  40. 
Lindner,  P.,  and  Stockhausen.     Wochenschr.  Brauerei  3,  No.  40  and  Bioch.  Centr. 
IV. 

3  Ehrlich,    F.      Uber    die    Spaltung   racemischer    Aminosauren   mittels  Hefe. 
Biochemische  Zeitschr.  8,  1908. 

4  Effront,   I.     Action  de  la  levure  de  biere  sur  les  acides  amides.     Comp. 
Rend.  Acad.  Sciences,  146,  1908. 

5  Pringsheim,    H.      Ueber   die   Stickstoffernahrung   der   Hefe.      Biochemische 
Zeitschrift,  3,  1907. 

6  Koser,  S.  A.  and  Rettger,  L.  F.     Studies  on  Bacterial  Metabolism.     The 
Utilization  of  Nitrogenous  Compounds  of  Definite  Chemical  Composition.     Jour. 
Inf.  Dis.  24  (1919)  301-321. 


NUTRITION  OF  YEASTS  73 

serve  bacteria  through  several  generations  while,  in  other  papers,  it  has 
been  shown  that  the  more  complex  split  products  of  protein  could  not. 

Jodin l  and  Hallier 2  attributed  to  yeasts  the  ability  to  fix  at- 
mospheric nitrogen.  Woff  and  Zimmermann,3  however,  could  not  con- 
firm these  statements.  Zikes 4  isolated  a  pseudo-yeast,  Torula  wiesner, 
to  which  he  attributed  the  ability  to  fix  atmospheric  nitrogen.  He 
isolated  this  organism  from  laurel  leaves,  and  secured  a  fixation  of 
2.3-2.4  mg.  per  gram  of  glucose.  Lohnis  and  Pillai5  secured  but 
slight  fixation  with  a  Torula.  With  Dematium  pullulans  there  was  a 
greater  fixation.  Lipman 6  found  that  yeasts  and  pseudo-yeasts 
could  fix  atmospheric  nitrogen.  The  action  went  better  in  solutions 
containing  dextrose.  Lindner  and  Naumann  7  could  secure  no  fixa- 
tion in  a  solution  containing  5  per  cent  of  dextrose,  .025  per  cent  of 
magnesium  sulfate,  0.5  per  cent  of  mono  potassium  phosphate  and  .025 
per  cent  of  asparagin.  Such  results  are  in  sharp  contrast  with  those 
of  other  investigators.  Kossowicz  8  reaches  the  conclusion  that  while 
yeasts  can  live  with  but  a  very  small  amount  of  nitrogen,  they  do  not 
have  the  power  to  fix  atmospheric  nitrogen.  Other  nitrogenous  com- 
pounds may  be  taken  from  the  air. 

Schwarz9  made  an  interesting  study  on  the  effect  of  adrenalin 
on  unicellular  organisms.  He  found  that  this  substance  acted  on 
organisms  without  nerves  just  as  it  did  on  higher  organisms.  Large 
quantities  of  sugars  were  used,  as  evidenced  by  much  C02.  The 
ability  to  utilize  non-diffusible  substances  was  acquired  (glycogen, 
casein,  alanine).  These  were  changed  to  fermentable  sugars.  Fur- 
ther experiments  with  glycogen,  starch,  alanine,  and  sodium  aspar- 
tate,  gave  CO2  when  adrenalin  was  present,  otherwise  not. 

Lindner10  studied  the  various  sources  from  which  a  yeast  could 

1  Jodin,  Compt.  rend.  Acad.  Sci.  55,  612. 

2  Hallier,  Zeit.  fur  Parasitenkunde.     1,  129. 

3  Woff  and  Zimmermann.    Jahresbericht  der  Ag.  Chemie,  13-15,  169. 

4  Zikes,  Sitzungsber.  Akad.  Wien.  mathe.  naturw.  Kl.  118,  1091. 
6  Lohnis,  F.  and  Pillai.    Cent.  Bakt.  Abt.  2,  20,  799. 

6  Lipman,  C.  B.    Nitrogen  fixation  by  yeasts  and  other  fungi.    J.  Biol.  Chem. 
10,  169-182. 

7  Lindner,  P.  and  Naumann,  C.  W.     Assimilation  of  nitrogen  of  air  by  yeasts 
and  molds.    Wochenschr.  Brau.  30,  58^-592. 

8  Kossowicz,    A.      Question   of   the   assimilation   of   elementary  nitrogen   by 
yeasts  and  mold  fungi.     Biochem.  Z.  64,  82-5.    Fixation  of  elementary  nitrogen 
by  saccharomycetes  (yeasts)  and  molds.    Z.  Garungsphysiologie,  5,  26. 

9  Schwarz,  O.     The  action  of  adrenalin  on  the  monocellular  organism.     Wien- 
klin.  Wochenschr.  24,  267-8.    Decomposition  of  nitrogenous  substances  by  yeasts. 
Biochem.  Z.  33,  30-1. 

10  Lindner,  P.     Results  obtained  in  fermentation  and  assimilation  experiments 
with  yeasts.    Chem.  Ztg.  34,  1144. 


74  PHYSIOLOGY  OF  YEASTS 

take  nitrogen.  Compounds  with  long  hydrocarbon  chains  were  easily 
assimilated.  The  ring  structures,  such  as  histidine,  were  used  with 
more  difficulty.  Leucine,  adenine,  and  lysine  were  easily  assimilated, 
but  thymine,  uracil,  choline,  hypoxanthine  more  difficultly.  Adenine, 
since  its  nitrogen  is  in  the  side  chain,  was  more  easily  assimilated 
than  hypoxanthine.  The  more  aerobic  yeasts  were  found  to  utilize 
more  difficultly  assimilable  nitrogen  more  easily. 

Zalesky  and  Israelsky  l  found  that  the  protein  content  of  yeast 
remained  constant  in  fermentation.  Asparagin  and  glutamic  acid 
support  synthesis  of  protein  while  glycocoll  and  phenylalanine  do  not. 

Thomas 2  and  Kolodziejska  3  found  two  new  proteins  in  yeasts. 
One  belonged  to  the  casein  group  and  the  other  to  the  vegetable  al- 
bumins. This  latter  was  named  cerevisin. 

Meisenheimer 4  studied  nitrogen  substance  in  yeast  by  autolysis 
in  presence  of  toluene.  All  of  the  common  amino  acids  were  found 
among  the  cleavage  products  of  yeast  protein.  Glucosamine,  so 
often  looked  for  in  vain,  was  demonstrated  to  be  present.  Nitrogen 
in  yeast  protein  is  distributed  as  follows: 

Ammonia  nitrogen 11  per  cent 

Alloxur  bases  (nuclein  bases)  nitrogen 7  per  cent 

Arginine-histidine  nitrogen 22  per  cent 

Lysine-choline  nitrogen 4  per  cent 

Monoamino  acid  nitrogen 56  per  cent 

Haydruck 5  has  shown  that  yeasts  are  suitable  foods  and  that 
they  should  be  looked  upon  favorably  as  constituents  in  the  human 
diet. 

Ehrlich  6  has  stated  that  amino  acids  are  deaminized  and  the  rest 
of  the  molecule  is  discharged  as  fatty  acid  or  alcohols.  Sugar  is  said 
to  be  the  sole  source  of  carbon.  To  secure  data  with  regard  to  what 
products  in  sugar  decomposition  went  to  make  up  the  complex  yeast 
proteins,  he  grew  Willia  anomala,  Hansen,  in  solutions  containing 
only  mineral  salts,  tyrosine  and  either  glycerol,  ethyl  alcohol,  methyl 

1  Zalesky,  W.  and  Israelsky,  W.     Synthesis  of  protein  in  yeast.     Ber.  deut. 
Bot.  Ges.  32  (1914),  472-9. 

2  Thomas,   P.     Protein  substances  of  yeast.     Comp.   rend.   Acad.  Sci.   156, 
2024-7. 

3  Thomas,  P.  and  Kolodziejska,  S.     Protein  matter  'of  yeast  and  its  products 
of  hydrolysis.    Comp.  rend.  Acad.  Sci.  157,  243-6. 

4  Meisenheimer,  J.     The  nitrogenous  substance  of  yeast.     Wochenschr.  Brau. 
32  (1915)  325-6. 

6  Haydruck,  F.  The  utilization  of  yeasts.  Brewers  Journal,  48,  57-58. 
1912. 

6  Ehrlich,  F.  The  formation  of  the  plasma  in  yeasts  and  molds.  Biochem. 
Z.  36,  447-97;  Chem.  Absts.  6  (1912)  240. 


HYDROCARBON  COMPOUNDS  75 

alcohol,  amyl  alcohol  or  lactic  acid.  Cultures  in  glycerol  and  ethyl 
alcohol  grew  as  well  as  the  control  in  sucrose.  Cultures  in  lactic  acid, 
methyl  alcohol  and  amyl  alcohol  grew  slightly,  and  formed  sufficient 
tyrosol  for  isolation.  Since  tyrosol  corresponding  to  nearly  all  of  the 
tyrosine  was  obtained,  it  shows  that  when  the  carbon  diet  is  limited 
to  simple  compounds,  the  yeast  does  not  utilize  the  carbon  of  amino 
acids. 

V 

3.  Hydrocarbon  Compounds 

The  yeasts,  being,  like  the  fungi,  without  chlorophyll,  are  not  able 
to  take  their  carbon  from  the  atmosphere.  They  have  to  resort  to 
other  compounds  as  sugars,  aldehydes,  acids,  etc. 

The  hydrocarbon  metabolism  of  yeasts  ought  to  be  looked  at  from 
two  standpoints.  One  should  distinguish  the  hydrocarbon  metabo- 
lism of  the  yeasts  during  the  aerobic  life,  that  is  the  plant-yeast, 
and  also  during  fermentation,  yeast-ferment.  In  the  two  cases,  they 
act  differently.  The  first  of  these  will  be  treated  here,  and  the  other 
when  we  take  up  alcoholic  fermentation. 

From  the  experiments  of  Laurent,1  it  is  evident  that  the  alcohols, 
aldehydes,  ethers,  fatty  acids,  amides,  glycocoll,  hydroquinone,  and 
cellulose  are  not  able  to  liberate  their  carbon  to  the  yeasts.  On  the 
contrary,  the  yeasts  are  able  to  take  it  from  acetates,  lactates,  cit- 
rates, tartrates,  malates,  succinates,  tartaric  acid,  malic  acid,  succinic 
acid,  lactic  acid,  glycerol,  from  sugars  of  the  C6Hi2O6  and  C^H^Ou 
series,  and  from  substances  capable  of  transforming  into  glucosides 
dextrine  lecithin,  asparagin,  peptones,  etc.  Bokorny  has  also  reached 
about  the  same  conclusion. 

It  seems  that  alcohol,  which  these  authors  regard  as  not  used  by 
yeasts,  is  able,  however,  to  be  used  by  certain  species.2  Thus  it  is 
that  recent  investigations  by  Trillat  and  Sauton,3  Kayser  and  Demo- 
Ion  4  indicate  that  they  oxidize  alcohol  to  the  aldehyde. 

The  investigations  of  Lindner  and   Saito  5  indicate  that  maltose 

1  Laurent,  E.     Nutrition  hydrocarbone"e  et  formation  du  glycogene  chez  la 
levure  de  biere.    Ann.  Past.  Inst.  3,  1889. 

2  We  shall  see  that  yeasts  are  able  to  live  for  many  years  in  liquids  which 
they  have  fermented.     It  is  probable  that  they  use  the  glycerol  and  succinic 
acid  (which  are  regarded  by  Laurent  as  being  able  to  supply  the  needs  of  yeasts 
for  carbon).    For  certain  species  alcohol  seems  to  be  the  source  of  carbon. 

3  Trillat  and  Sauton.     L'aldehyde  ace"tique  est-il  un  produit  normal  de  la 
fermentation  alcoolique?    Ann.  Inst.  Past.  24.     1910. 

4  Kayser  and  Demolon.    Sur  la  vie  de  la  levure  apres  la  fermentation  alcooli- 
que.   Comp.  Rend.  Acad.  Sci.  149.     1909. 

5  Lindner,   P.   and  Saito,   K.     Assimilierbarkeit  verschiedener    Kohlehydrate 
Hefe.    Woch.  Brauerei  No.  41.     1910. 


76  PHYSIOLOGY  OF  YEASTS     . 

is  best  adapted  to  yeast  metabolism.  This  sugar  is  assimilated  by 
practically  all  of  the  yeasts.  Dextrine  is  transformed  by  certain 
varieties  (Mycoderma  and  Torula),  but  is  not  well  adapted  to  others. 
Sucrose  which  is  so  easily  fermented  does  not  play  any  role  in  as- 
similation. The  same  is  true  with  regard  to  glucose,  levulose,  raffi- 
nose,  and  arabinose.  Finally,  lactose  is  not  assimilated  except  in 
isolated  cases.  Lindner,1  in  a  later  publication,  stated  that  maltose 
is  easily  available  as  a  nutrient  sugar,  glucose,  fructose,  and  cane  sugar 
being  less  valuable.  In  fact  sucrose  was  often  valueless. 

On  the  other  hand,  the  experiments  of  these  authors  have  showed 
that  there  is  no  relation  between  the  fermentability  of  a  sugar  and  its 
use  as  an  nutrient.  Thus  it  is  that  one  frequently  meets  yeasts 
which,  in  functioning  aerobically,  energetically  assimilate  a  sugar, 
while,  functioning  anaerobically,  they  are  unable  to  ferment  it.  One 
may  encounter,  although  rarely,  a  yeast  which  is  able  to  ferment  a 
sugar  and  not  able  to  use  it  as  a  nutrient.  Such  is  the  case  with 
S.  Ludwigii,  exiguus,  cartilaginosus  and  Sch.  Pombe  and  mellacei 
which  produce  an  active  fermentation  of  glucose,  levulose  and  sac- 
charose but  are  unable  to  assimilate  any  of  them. 

Kluyver 2  attributed  the  statements  that  yeast  is  able  to  assimilate 
maltose  to  the  fact  that  the  maltose  contained  glucose.  When  the 
maltose  was  purified  and  freed  from  the  glucose  no  assimilation  was 
secured.  Lindner  3  has  shown  that  maltose  is  easily  assimilated  by 
yeasts.  Glucose,  sucrose,  and  fructose  were  less  satisfactory. 

It  is  known  since  the  work  of  Errera  that  glycogen  is  abundant 
in  yeast  cells.  Since  it  is  there  so  abundantly,  it  seems  to  have  con- 
siderable importance  in  the  life  of  the  yeasts.  The  study  of  the  con- 
ditions for  its  formation  is  very  interesting  and  may  explain  many 
facts  with  regard  to  the  hydrocarbon  nutrition  of  yeasts.  This  study 
has  been  made  by  Laurent  who  has  stated  that  glycogen  is  able  to 
be  formed  at  the  expense  of  the  following  substances: 

Lactates 

Succinic  acid  and  ammonium  succinate 

Malic  acid  and  malates 

Mannite 

Sugars  of  the  C6Hi2O6  and  Ci2H22On  series 

Glycogen 

1  Lindner,  P.     The  results  obtained  in  fermentation  and  assimilation  experi- 
ments with  yeasts.    Chem.  Ztg.  34,  1144.    Chem.  Absts.  6  (1912)  1050. 

2  Kluyver,  A.  J.     Assimilability  of  maltose  by  yeasts.     Biochem.  Zeit.  52, 
486-493. 

3  Lindner,  P.     The  results  obtained  in  fermentation  and  assimilation  experi- 
ments with  yeasts.    Chem.  Ztg.  34,  1144. 


HYDROCARBON  COMPOUNDS  77 

Gum  arable 

Erythrodextrine  and  dextrine 

Mucic  acid 

Asparagin  and  glutanine 

Salicine,  amygdalin,  and  other  glucosides 

Egg  albumin 

Peptones  from  fibrine  and  casein. 

It  is  stated,  according  to  Laurent,  that  glycogen  is  able  to  serve 
in  the  hydrocarbon  nutrition  of  yeasts  and  the  production  of  glyco- 
gen in  the  cell.  This  statement  is  without  doubt  in  error.  From  the 
investigations  of  Koch  and  Hosaeus,1  it  has  been  established  that 
glycogen  is  not  absorbed  by  yeasts,  for  it  is  not  diffusible  through  the 
cell  membrane  any  more  than  the  glycogenase  enclosed  in  the  cell. 

The  conclusions  of  Laurent  have  been  confirmed,  in  most  part  by 
Cremer.  This  author  has  also  stated  that  yeasts  deprived  of  their 
glycogen  by  autofermentation  phenomena,  which  we  shall  study 
further,  produce  it  after  a  few  hours  if  they  are  placed  in  a  solution 
with  sugar  (saccharose,  levulose,  glucose,  d-galactoce,  d-mannose) 
but  not  if  furnished  with  arabinose,  rhamnose,  sorbose,  lactose,  glyc- 
erol  or  glycogen. 

According  to  Laurent,  Boullinger,  and  Kayser  and  Meissner, 
glycogen  is  rare  or  completely  absent  at  the  beginning  of  fermentation; 
it  increases  progressively  and  soon  reaches  a  maximum.  It  disappears 
at  the  end  of  fermentation.  These  results  are  absolutely  confirmed 
by  those  of  Wagner,  Kohl  and  Guilliermond.  It  seems  that  toward 
the  middle  of  the  fermentation,  glycogen  accumulates  in  the  cell 
much  more  quickly  than  it  is  consumed. 

The  investigations  of  Lindner  and  Will  indicate  that  glycogen  is 
unevenly  distributed  in  the  yeast  cell  and  is  able  to  exist  under  very 
variable  conditions.  Thus  it  is  that  Lindner  has  observed  glycogen 
in  yeasts  which  had  been  cultivated  on  gelatin  for  4  months.  The 
same  is  true  of  most  reserve  products.  It  is  difficult  to  state  accu- 
rately the  conditions  under  which  the  formation  of  glycogen  is  greater 
than  the  expenditure. 

These  authors  have  come  to  regard  glycogen  as  a  transitory  sub- 
stance in  the  cell  and  intermediary  between  the  sugars  and  alcohols. 
According  to  Griiss,  it  constitutes,  as  we  shall  show  later  on,  an  ex- 
clusive substance  destined  for  respiration  (in  presence  of  air)  and  for 
fermentation  (in  the  absence  of  air).  Glycogen  is  formed  from  the 
sugars  which  are  dissolved  by  the  cell  and  is  transformed  either  into 

1  Koch,  A.  and  Hosaeus.  Ueber  einen  neuen  Froschlauch  der  Zuckerfabriken. 
Cent.  Bakt.  16,  1894. 


78  PHYSIOLOGY  OF  YEASTS 

carbonic  acid  and  water  in  aerobic  life,  or  into  carbonic  acid  and 
alcohol  in  anaerobic  life. 

Kohl  holds  the  same  opinion  based  on  the  following:  Glycogen 
is  especially  abundant  in  yeast  cells  during  active  fermentation.  It 
is  not  found  in  cells  about  to  sporulate  or  in  ascospores.  Our  ob- 
servations have  shown  on  the  contrary  that  glycogen  is  very  abun- 
dant, not  only  during  fermentation  but  also  during  the  formation  of 
ascospores  in  the  course  of  their  maturation.  Part  is  used  in  the 
formation  of  the  ascospore  while  the  rest  is  kept  in  reserve  for  their 
germination.  The  investigations  of  Will  have  shown  that  the  durable 
cells  contain  large  quantities  of  glycogen.  These  facts  do  not  exclude 
the  theory  of  Grtiss,  for  it  is  possible  that  glycogen  is  a  reserve  prod- 
uct especially  for  respiration. 

Henneberg  l  states  that  glycogen  may  occur  in  both  normal  and 
abnormal  yeast.  Since  yeast  cells  containing  more  that  53  per  cent  of 
protein  usually  contain  little  glyc.ogen,  it  is  probable  that  yeast  cells 
in  potato  mashes,  etc.,  will  contain  little.  Bruschi 2  found  that 
antiseptics,  such  as  chloroform,  ether,  thymol  and  formalin,  did  not 
completely  stop  the  formation  of  glycogen  even  though  fermentation 
was  impeded.  The  production  of  alcohol  determined  the  amount  of 
glycogen  formed.  It  is  stated  that  glycogen  is  formed  by  the  con- 
densation of  some  intermediate  product  of  fermentation.  Kullberg  3 
reported  an  inverse  relation  between  the  nitrogen  content  of  the  yeast 
cell  and  the  glycogen  content. 

Will  and  Heuse 4  found  that  ethylacetate  satisfied  the  carbon 
requirements  of  yeast  and  that  they  could  grow  without  the  presence 
of  organic  matter.  Lindner  and  Cziser6  found  alcohol  a  source  of 

carbon.       Stockhausen 6   confirmed   this   opinion.      Lindner 7  has   re- 

i 

1  Henneberg,  W.    Amount  of  glycogen  in  differently  fed  yeast  cultures.    Bieder- 
mann's  Zentr.     1912.     277-8;   Chem.  Absts.  6,  1917-18.     1912. 

2  Bruschi,  D.     Formation  of  glycogen  in  the  yeast  cell.     Att.  accad.  Lincei, 
21, 1,  54-flO;   Chem.  Absts.  6,  1018-19,  1912;    Cent.  Bakt.  Abt.  II,  35,  316;    Chem. 
Absts.  7  (1913)  495. 

3  Kullberg,   S.     Simultaneous  change  in  the   content  of  glycogen,    nitrogen 
and  enzyme  in  living  yeast.     Zeit.  physiol.  Chem.  92,  340-359.     (1914);    Chem. 
Absts.  9  (1915),  471. 

4  Will,  H.,  and  Heuse,  R.     Ethyl  acetate  as  a  source  of  carbon  for  yeasts 
and  other  budding  fungi.     Zeit.  ges.  Brauw.  35,  128-9,  Chem.  Absts.  6  (1912) 
1626. 

6  Lindner,  P.  and  Cziser.  Alcohol,  a  more  or  less  excellent  nutrient  medium 
for  different  organisms.  Wochenschr.  Brau.  29,  1-6;  Chem.  Absts.  6  (1912)  1916. 

6  Stockhausen,   F.     Alcohol  assimilation  by  yeasts.     Chem.  Ztg.   35,    1197. 
Chem.  Absts.  6  (1912)  3106. 

7  Lindner,  P.     Non-assimilation  of  methyl  alcohol  by  microorganisms  capable 
of  assimilating  ethyl  alcohol.     2.  Spiritusind.  35,  185;  Chem.  Absts.  6  (1912)  1917, 


HYDROCARBON  COMPOUNDS  79 

ported  that  S.  membranaefadens  could  take  its  carbon  from  ethyl  but 
not  from  methyl  alcohol.  Bokorny  l  studied  the  sources  of  carbon 
for  yeasts  and  stated  that  urea  cannot  supply  carbon  for  yeasts.  This 
is  confirmed  by  Lindner  and  Wiist 2  who  found  that  urea  could  supply 
nitrogen  but  not  carbon.  Pentoses  were  found  by  Bokorny  to  be  not 
fermented  but  could  serve  as  a  source  for  carbon.  Others  have  shown 
that  different  organic  acids,  glycerol,  asparagin,  peptone,  etc.,  could 
be  used.  Will 3  found  that,  in  mineral  media,  esters  could  act  as  a 
source  of  carbon.  Lindner4  studied  the  assimilability  of  the  various 
carbohydrates.  He  investigated  dextrose,  mannose,  galactose,  levulose, 
trehalose,  sucrose,  maltose,  lactose,  melibiose,  raffinose,  a-methyl- 
glucoside,  xylose  and  rhamnose.  The  simple  sugars,  trehalose,  suc- 
rose and  maltose,  were  fermented.  Lactose  was  not.  In  certain  cases, 
there  was  a  questionable  fermentation  of  xylose  and  rhamnose.  Meli- 
biose was  not  fermented  by  top  yeast.  There  seemed  to  be  a  marked 
difference  in  action  toward  a-methylglucoside.  In  a  later  paper 
Lindner  5  again  found  maltose  better  than  other  sugars. 

Bokorny  5  has  stated  that  external  factors  have  great  influence  in 
fermentation  and  assimilation  studies.  Light  is  not  important  for 
the  yeasts  but  plenty  of  air  is  important.  The  increase  in  dry  sub- 
stance in  this  experiment  was  taken  as  the  criterion  of  assimilation. 
Assimilation  was  promoted  by  free  KOH  at  certain  concentrations. 

Kita  7  found  that  purified  maltose  was  less  easily  assimilated  than 
unpurified.  He  attributed  this  to  the  fact  that  in  the  impure  maltose 
there  was  an  oryzanin-like  compound.  The  same  thing  was  observed 
by  Kluyver.8 

1  Bokorny,  T.     The  formation  of  protein  from  different  sources  of  carbon. 
Munch,  med.  Wochenschr.  63,  791-2;    Chem.  Absts.  11  (1917)  2813. 

2  Lindner,  P.  and  Wiist,  G.     Wochenschr.  Brau.  30,  477-9.     Chem.  Absts.  8 
(1914)  727. 

3  Will,   H.     Influence  of  esters  on  yeasts  and  other  budding  fungi.     Cent. 
Bakt.  Abt.  II.  38,  539-76.    Chem.  Absts.  8  (1914)  357. 

4  Lindner,  P.     Assimilation  of  various  carbohydrates  by  different  yeasts  and 
the  like.    Wochenschr.  Brau.  47,  561-563.    Chem.  Absts.  6  (1912)  1808. 

Lindner,  P.  and  Saito,  K.  The  assimilation  of  various  carbohydrates  by 
different  yeasts.  Wochenschr.  Brau.  27,  509-13;  Chem.  Absts.  II,  476,  5  (1911) 
1489. 

5  Lindner,  P.     The  results  obtained  in  fermentation  and  assimilation  experi 
ments  with  yeasts.    Chem.  Ztg.  34,  1144. 

6  Bokorny,  Th.      Relation  between  sugar  fermentations  and  sugar  assimila- 
tion.   Allgem.  Brau-Hopfen  Ztg.  57,  447-80;   Chem.  Absts.  12  (1918)  847. 

7  Kita,  G.     The  question  of  the  assimilability  of  maltose  by  yeasts.     Zeit. 
Garungsphysiologie,  4,  231-4.    Chem.  Absts.  8  (1914)  3809. 

8  Kluyver,  A.  J.     Assimilability  of  maltose  by  yeasts.     Biochem.  Z.  52,  486; 
Chem.  Absts  8  (1914)  359. 


80  PHYSIOLOGY  OF  YEASTS 

Lindner,1  using  Saccharomyces  membranaefaciens,  which  assimilates 
ethyl  alcohol  in  the  absence  of  other  sources  of  carbon,  could  not  find 
an  assimilation  of  methyl  alcohol. 

Stockhausen  2  inoculated  a  mineral  nutrient  solution  containing 
(NH4)2SO4  as  the  source  of  nitrogen  and  4  per  cent  of  alcohol  as  the 
source  of  carbon.  Excellent  yeast  growth  was  secured  in  a  few  days. 
This  author  argues  that  alcohol,  in  this  case,  was  a  food  from  which 
plasma,  cell  membrane  and  fat  could  be  built.  The  same  fact  was 
established  by  Lindner  and  Czier.3 

Rubner 4  believes  that  other  than  physical  conditions  control 
assimilation  and  nourishment  of  yeasts  since  they  take  what  sugar  is 
needed  irrespective  of  its  concentration.  Rubner  found  that  live 
yeast,  as  well  as  yeast  killed  with  toluene,  quickly  took  up  sugar 
from  a  solution  without  fermentation,  while  yeast  heated  to "100°  C. 
did  not.  This  author  regards  the  yeasts  as  organisms  possessing  great 
energy  transformations  per  unit  mass.  Lindner  5  found  that  growth 
took  place  at  the  expense  of  atmospheric  nitrogen.  Ethyl  alcohol 
and  free  ammonia  were  used  to  build  protoplasm.  Saccharomyces 
acetethylicus  assimilated  nitrogen  from  nitrates.  Urea  provided  assimi- 
lated nitrogen  particularly  if  maltose  was  present.  Maltose  was  found 
to  be  the  best  source  of  carbon.  Melibiose  and  ramnose  are  readily 
assimilated  by  yeasts  even  by  some  which  do  not  ferment  them. 


Respiration 

We  have  seen  that  yeasts  placed  in  contact  with  air,  act  like 
ordinary  plants  without  causing  alcoholic  fermentation.  Like  all 
living  matter,  they  respire;  they  take  in  oxygen  and  liberate  carbon 
dioxide.  The  investigations  of  Schutzemberger,  Grehant  and  Quin- 
quand  6  have  shown  that  they  are  very  eager  for  oxygen. 

Schutzemberger  has  stated  that  fresh  yeast  put  into  water  well 
aerated  at  fermentation  temperature  is  obliged  to  live  at  the  expense 

1  Lindner,  P.    Non-assimilability  of  methyl  alcohol  by  microorganisms  capable 
of  assimilating  ethyl  alcohol.     Zeit.  Spiritusind.  35,  185;     Chemical  Abstracts,  6 
(1912)  1917. 

2  Stockhausen,   F.     Alcohol  assimilation  by  yeasts.     Chem.  Ztg.   35,    1197. 
Chem.  Absts.  9  (1912)  4106. 

3  Lindner,  P.  and  Czier,  S.    Alcohol  a  more  or  less  excellent  nutrient  medium 
for  different  organisms.    Wochenschr.   Brau.   29,   1-6;   Chem.  Absts.  II,   476,   6 
(1912),  1916. 

4  Rubner,  M.     Assimilation  of  nourishment  by  the  yeast  cells.     Cent.  Bakt. 
Abt.  II.  38  (1914),  128.     Chem.  Absts.  9  (1915),  93. 

5  Lindner,  P.     Results  of  recent  experiments  on  assimilation  by  yeasts  and 
molds.    Zeit.  angewandte  Chemie,  25,  1175.     Chem.  Abstracts  7  (1913)  2055. 

6  Grehant  and  Quinquand.    Ann.  des.  Sc.  nat.  Bot.  1889. 


ALCOHOLIC   FERMENTATION  81 

of  its  reserve  products,  absorbing  oxygen  and  giving  off  carbon  dioxide. 
Yeasts  are  also  able  to  take  oxygen  from  compounds  in  which  it  is 
loosely  combined.  It  is  thus,  as  Schutzemberger  and  Risler  have 
stated,  that  when  fresh  yeast  is  placed  in  arterial  blood  or  in  a  solu- 
tion of  hemoglobin  saturated  with  oxygen,  the  color  passes  from  a 
deep  red  to  a  bluish  black.  In  this  case  the  yeasts  take  their  oxygen 
from  the  blood  and  act  like  tissue  cells  in  the  animal  body.  While 
yeasts  are  able  to  take  oxygen  only  from  such  unstable  combinations 
as  hemoglobin,  they  are  not  able  to  get  it  from  compounds  which 
hold  it  firmly.  For  instance,  they  are  without  action  on  indigo  carmin 
which  some  bacteria  decolorize  so  strongly. 

The  respiratory  activity  measured  by  the  oxygen  consumed  in  a 
unit  time  by  a  unit  weight  of  yeast,  varies  with  the  temperature. 
It  is  very  feeble  at  10°,  increases  slowly  up  to  18°,  and  attains  its 
maximum  towards  60°.  It  falls  quickly  after  the  death  of  the  yeast. 
The  experiments  of  Grehant  and  Quinquand  have  given  the  same  re- 
sults. They  have  shown  that  respiration  diminishes  a  little  and  re- 
duces itself  to  a  minimum  during  the  anaerobic  life  of  the  yeast, 
but  never  totally  disappears.  As  has  been  stated  before,  Griiss  re- 
garded glycogen  as  important  in  respiration.  According  to  this  au- 
thor, glycogen  is  a  reserve  product  utilized  in  respiration  and  fermen- 
tation.- It  is  by  means  of  their  oxidases  that  yeasts  oxidize  the  glucose 
secured  by  hydrolysis  of  glycogen,  transforming  it  into  carbon  clioxide 
and  water. 

ALCOHOLIC  FERMENTATION 

General  Characteristics  of  Alcoholic  Fermentation 

Conditions  Necessary  for  Its  Production 

While  the  molds  produce  very  quickly  on  the  surface  of  liquids 
a  vigorous  vegetation,  and  thus  live  in  contact  with  air,  the  greater 
number  of  the  yeasts  develop  at  the  bottom  of  culture  media  in  the 
form  of  a  sediment,  and  it  is  only  under  exceptional  circumstances 
that  they  develop  on  the  surface  in  the  form  of  a  pellicle  often 
called  a  scum. 

When  cultivated  in  a  dish  containing  sugar  solution  in  a  thin 
layer,  the  supply  of  air  is  sufficient  to  allow  a  vigorous  growth  of 
the  yeast.  Under  these  circumstances,  it  decomposes  the  sugar, 
using  part  for  maintaining  protoplasm  or  constructing  new  substance, 
and  transforming  the  rest  by  oxidation  to  carbon  dioxide  and  water. 
In  a  word,  it  is  aerobic  and  acts  like  other  plants. 

The  activities  are  different  when  it  is  put  into  a  flask  almost 
completely  filled  with  a  sugar  solution  and  to  which  air  does  not 


82  PHYSIOLOGY  OF   YEASTS 

have  access.  The  yeast  grows  at  the  bottom  of  the  flask  and  finds 
a  bad  supply  of  oxygen.  In  this  case  it  uses  little  sugar  for  main- 
taining itself  and  scarcely  multiplies;  the  rest  of  the  sugar  is  changed 
into  alcohol  and  carbon  dioxide  by  the  enzymes. 

In  anaerobic  life  the  yeasts  are  not  able  to  secure  their  energy 
by  oxidation.  Quite  another  chemical  change  is  involved;  this 
is  the  enzymatic  change  of  sugar  into  alcohol  and  carbon  dioxide. 
One  easily  conceives  that  much  less  energy  is  secured  by  this  proc- 
ess than  by  an  oxidation.  Much  of  the  energy  will  be  used  for 
building  up  a  very  small  quantity  of  new  protoplasm.  The  trans- 
formation of  sugar  into  alcohol  will  be  considerable  for  a  minimum 
growth  of  the  yeast.  The  memorable  researches  of  Pasteur  l  have 
enriched  our  information  with  regard  to  this  change.  This  illus- 
trious savant,  by  a  series  of  experiments,  demonstrated  that  the 
scarcer  the  amount  of  oxygen,  the  greater  was  the  amount  of  fermen- 
tation. 

The  best  means  of  propagating  a  yeast  under  aerobic  conditions  is, 
as  we  have  stated  above,  growing  it  in  a  shallow  dish  with  a  few 
centimeters  of  nutrient  medium.  Under  such  conditions,  Pasteur, 
at  the  end  of  24  hours,  has  obtained  24  milligrams  of  yeast  cells 
with  a  consumption  of  98  milligrams  of  sugar.  No  trace  of  alcohol 
is  found  in  the  medium.  At  the  end  of  48  hours,  Pasteur  obtained 
127  milligrams  of  yeast  cells  for  1.04  grams  of  sugar  decomposed. 
The  yeast  was  almost  exclusively  an  agent  of  oxidation  and  acted 
like  other  plants.  It  consumed  a  large  part  of  the  sugar  for  its 
maintenance  and  multiplication  and  its  weight  increased  in  a  con- 
siderable proportion. 

It  does  not  act  like  this  when  put  into  a  flask  to  which  air  does 
not  have  free  access.  For  10  grams  of  sugar  decomposed  Pasteur 
secured  only  0.44  gram  of  yeast  cells.  The  yeast  had  scarcely  mul- 
tiplied; on  the  contrary,  the  proportion  of  sugar  changed  to  alcohol 
became  greater. 

Pasteur  has  continued  his  experiments  by  putting  a  thin  layer 
of  liquid  into  the  same  flask.  This  time,  the  fermentation  was  longer 
and  the  weight  of  the  yeast  less  perceptible;  but  the  same  propor- 
tion of  alcohol  is  found  as  in  the  alcohol  fermentations  so-called. 
In  this  last  experiment,  the  liquid  in  the  flask  was  aerated,  retaining 
a  small  quantity  of  oxygen  which  the  yeast  utilized  at  the  beginning 
of  its  development.  On  the  other  hand,  the  yeast  may  come  from 
cultures  which  are  in  contact  with  air;  the  cells  then  have  been  able 

1  Pasteur.  Memoire  sur  la  fermentation  alcoolique.  Ann.  de  Ghim.  et  do 
phys.  1859:  Influence  de  Toxygene  sur  le  de"v.  de  la  levure  et  de  la  ferm.  ale. 
Bull.  Soc.  Chim.  1861. 


ALCOHOLIC   FERMENTATION  83 

to  accumulate  sufficient  oxygen.  Pasteur  also  repeated  the  experi- 
ment by  eliminating  these  two  sources  of  oxygen.  For  this  he  inoc- 
ulated a  trace  of  the  yeast  taken  at  the  end  of  a  fermentation  and  which 
had  not  been  in  contact  with  air,  into  a  flask  almost  completely  filled 
with  sugar  solution  which  had  been  boiled.  Under  such  conditions, 
the  fermentation  was  very  slow.  Pasteur  made  it  endure  three  months. 
At  the  end  of  this  time,  45  grams  of  sugar  had  disappeared  and  only 
0.255  gram  of  yeast  had  been  formed.  The  yeast,  then,  did  not 
develop.  Thus  from  these  experiments  one  sees  the  weight  of  sugar 
which  a  unit  weight  of  yeast  is  able  to  decompose  into  alcohol  and 
carbonic  acid  and  at  the  same  time  diminish  the  activity  of  life  and 
the  power  of  reproduction. 

All  of  this  demonstrates  that  fermentation  is  correlated  with 
anaerobic  development  and  that  it  is  more  active  when  oxygen  is 
absent.  In  the  presence  of  air,  the  yeast  functions  like  a  plant. 
It  is  nourished,  respires  and  multiples.  When  placed  in  a  reduced 
air  supply,  it  gives  up  or  suppresses  almost  completely  its  multi- 
plication. From  the  alcoholic  fermentation,  it  draws  the  energy 
which  it  needs.  Then,  the  scarcer  the  oxygen,  the  slower  the  multi- 
plication. 

The  experiments  of  one  of  Pasteur's  students,  Denys  Cochin, 
indicate  that  fermentation  does  not  take  place  in  the  total  absence 
of  oxygen.  The  yeasts  are  not  strict  anaerobic  organisms  but  are 
intermediate  between  the  aerobes  and  anaerobes.  It  is  necessary 
for  them  to  always  have  a  little  oxygen,  and  it  may  be  said  that  every 
yeast  that  does  not  receive  a  minimum  supply  of  oxygen  from  its 
ancestors  or  does  not  find  it  in  the  culture  medium,  will  perish. 
Oxygen  seems  to  have  a  beneficial  action  on  the  cellular  activity  and 
the  secretion  of  enzymes. 

These  results  have  been  contradicted  to  show  that  fermentation 
though  favored  by  the  absence  of  oxygen,  is,  moreover,  accomplished 
in  the  scarcity  of  air  (Brefeld,  Hansen,  Wehmer),  but  any  invali- 
dation of  Pasteur's  results  seems  not  to  have  been  produced  up  to  the 
present  time.  More  recently  Palladine  and  Iraklionoff  1  have  shown 
that  if  a  yeast  is  able  to  produce  small  quantities  of  alcohol,  even 
in  the  presence  of  air,  it  may  be  explained  by  assuming  the  presence 
of  peroxidases  which  reduce  peroxides,  freeing  nascent  oxygen  which 
may  be  active. 

1  Palladine,  W.,  and  Iraklionoff,  P.  La  peroxydase  et  les  pigments  respira- 
toires  chez  les  plantes.  Rev.  gen.  de  Bot.  23.  1911. 


84  PHYSIOLOGY  OF  YEASTS 


Prevalence  of  Alcoholic  Fermentation 

The  phenomenon  of  alcoholic  fermentation  is  not  limited  to  the 
yeasts.  Many  of  the  molds  l  are  also  able  to  ferment  the  sugars. 
But  this  fermentation  is  less  active  and  is  more  prolonged.  Further- 
more, it  is  only  produced  under  certain  conditions.  A  few  examples 
taken  from  DuClaux  will  be  given. 

Among  the  molds,  it  is  known  that  Sterigmatocystis  nigra,  which  is 
exclusively  aerobic,  never  produces  alcoholic  fermentation.  Some  of 
the  other  species,  such  as  Aspergillus  glaucus  and  Penidllium  glau- 
cum,  are  able  to  cause  a  slight  fermentation.  If,  for  example,  some 
of  the  conidia  of  Penidllium  glaucum  are  inoculated  into  a  Pasteur 
flask  containing  a  sugar  medium,  a  well-developed  mycelium  is  pro- 
duced on  the  surface;  after  a  time,  the  air  becomes  reduced  in  con- 
centration and  carbon  dioxide  accumulates  with  traces  of  alcohol. 
The  amount  of  alcohol  will  always  remain  very  small,  and  will  scarcely 
pass  from  1000th  to  1500th  of  the  total  volume.  Under  the  same 
conditions,  Aspergillus  glaucum  will  produce  large  amounts  of  alcohol. 
Pasteur  has  shown  that  in  a  culture  of  Aspergillus  glaucum  cultivated 
in  122  c.c.  of  beer  wort  for  a  year,  4.4  c.c.  of  alcohol  were  produced 
by  a  weight  of  yeast  which  scarcely  surpassed  0.5  gram  in  the  dry 
state.  About  seven  times  the  weight  of  the  plant  in  alcohol  were 
produced. 

Many  other  molds  possess  a  fermenting  action.  Mucor  racemosus 
will  produce  under  the  same  conditions  more  alcohol  than  the  two 
fungi  mentioned  above.  According  to  Pasteur,* the  weight  of  alcohol 
will  be  10  or  20  times  the  weight  of  the  mycelium.  Mucor  mucedo,  dr- 
dnelloides  and  erectus,  Amylomyces  rouxii  and  Aspergillus  oryzae 
are  in  the  same  category.  With  the  molds,  however,  fermentation 
requires  a  longer  time  than  the  yeasts  require  to  ferment  the  same 
amount  of  sugar. 

Comparison  of  Intramolecular  Respiration  with 
Alcoholic  Fermentation 

As  Pasteur  has  pointed  out,  alcoholic  fermentation  is  not  limited 
to  the  molds  nor  to  the  yeasts,  but  is  carried  on  in  all  living  cells 
which  contain  sugar.  Indeed,  alcoholic  fermentation  ought  to  be 
compared  to  what  is  called  "intramolecular  respiration." 

Berard  demonstrated,  for  the  first  time  in  1821,  that  fruits  which 
were  exposed  to  the  sun  absorbed  oxygen  and  liberated  carbon  dioxide; 

4  Certain  bacteria  are  also  known  which  produce  alcoholic  fermentation. 


PREVALENCE  OF  ALCOHOLIC   FERMENTATION       85 

in  a  word,  they  respired.  But  if  placed  in  an  atmosphere  of  limited 
oxygen  supply,  they  quickly  absorb  this  and  continue  to  liberate  car- 
bon dioxide.  Lechartier  and  Bellamy  (1869)  established  the  formation 
of  alcohol  under  these  conditions.  This  phenomenon  remained  un- 
explained until  Pasteur  undertook  his  experiments,  when  it  was 
definitely  proven  to  be  an  alcoholic  fermentation.  Pasteur  placed 
plums  under  a  flask  filled  with  CO2  and  secured  6  grams  of  alcohol 
after  8  days.  This  same  experiment  was  repeated  on  other  tissues 
containing  sugar.  Muntz  has  been  able  to  form  alcohol  by  placing 
some  of  the  higher  fungi  in  a  sugar  solution.  Maze,  Goldewsky  and 
Polszeniuz,  and  a  few  other  authors,  have  secured  similar  results 
with  certain  plants.  Green  peas  have  the  property,  when  placed  under 
water  away  from  air,  of  causing  a  sugar  solution  to  ferment  by  simple 
contact,  and  act  exactly  as  the  yeasts  except  with  less  activity. 

It  seems,  then,  as  if  all  cells  which  contain  sugar  are  able,  in 
the  absence  of  oxygen,  to  function  as  yeast  cells  and  produce  alco- 
holic fermentation.  We  shall  see  that  the  yeasts,  themselves,  during 
inanition  are  able  to  produce  a  fermentation  of  their  reserve  glycogen, 
thus  causing  a  sort  of  autofermentation. 

"  The  alcoholic  fermentation  is  not  a  characteristic  inherent  alone 
in  the  yeast  cell  nor  a  necessary  manifestation  for  its  existence.  It 
is  a  characteristic  variable  with  the  conditions,  but  rather  general. 
The  yeasts  differ  from  other  plants  only  in  the  characteristic  that 
they  are  able  to  adapt  themselves  better  to  anaerobic  life  and  thus 
show  this  new  phenomenon  which  is  of  so  much  industrial  impor- 
tance." 

Differences  in  the  Fermenting  Function  in  Different  Yeasts 

If  alcoholic  fermentation  is  not  a  function  special  to  the  yeasts, 
one  must  not  regard  it  as  a  specific  characteristic.  Many  of  the 
yeasts  are  not  able  to  produce  alcoholic  fermentation  but  act  only 
as  oxidizing  agents,  as  aerobes.  Such  are  all  of  the  Mycoderma  and 
even  the  true  yeasts  producing  endospores,  as  Pichia  hyalospora. 
These  form  a  luxuriant  veil  at  the  beginning  of  their  development  on 
carbohydrate  media,  which  covers  the  surface  of  the  liquid;  they  live 
then  in  contact  with  oxygen,  consuming  this  gas  and  liberating  carbon 
dioxide.  Many  of  the  Torula,  although  vegetating  at  the  bottom  of 
liquids,  are  also  in  the  same  class.  Some  of  the  other  yeasts  act  like 
molds  which  we  have  just  discussed.  They  live  by  preference  in  con- 
tact with  air  and  possess  only  mediocre  fermenting  capacity.  For 
example,  the  Willia  and  Pichia  and  the  myco-yeast  of  Duclaux  are 
such. 

This  yeast  develops  on  liquid  media  with  a  typical  veil  or  scum 


86  PHYSIOLOGY  OF  YEASTS 

which  is  folded  in  limited  space  and  may  become  rather  thick.  In 
such  a  form  the  myco-yeast  is  a  strong  oxidizing  agent  and  acts  like 
an  aerobic  mold.  It  does  not  produce  alcohol.  If  the  veil  or  pellicle 
is  transferred  to  a  flask  filled  to  the  neck  with  a  carbohydrate  me- 
dium, an  alcoholic  fermentation  results.  From  this  moment,  the 
myco-yeast  acts  like  a  yeast  enzyme;  its  development  is  slow  and 
almost  the  same  weight  is  maintained  which  it  had  at  the  beginning. 
This  yeast  does  not  produce  as  much  alcohol  as  ordinary  yeasts. 
It  never  exceeds  3  per  cent.  Monilia  Candida,  a  fungus,  intermediate 
between  the  yeasts  and  molds,  acts  in  the  same  way.  It  produces 
a  veil  on  the  surface  of  the  medium  and  grows  aerobically;  at  the 
bottom  of  the  flask  it  may  appear  as  a  deposit  which  decomposes 
the  sugar.  Hansen  found  that  it  yielded  1.1  per  cent  of  alcohol  in 
the  time  interval  in  which  S.  cerevisiae  would  yield  6  per  cent. 

Excepting  these  species,  most  yeasts,  especially  the  industrial 
yeasts,  are  very  energetic  agents  in  alcoholic  fermentation.  These 
are  distinguished  from  the  myco-yeast  and  Monilia  Candida  by  the 
fact  that  they  vegetate  almost  solely  at  the  bottom  of  the  culture 
flasks  and  form  no  veil  at  the  surface.  They  almost  always  grow 
under  conditions  of  restricted  aeration  and  possess  the  ability  to 
adapt  themselves  to  anaerobic  life  which  distinguishes  them  from 
other  plants.  These  yeasts  may  then  be  regarded  as  true  agents  of 
alcoholic  fermentation.  We  have  stated  above  that  the  industrial 
yeasts  may  form  about  six  times  as  much  alcohol  as  the  intermediate 
forms. 

Fermentable  Sugars 

It  is  to  the  renowned  researches  of  Fischer  and  Thierfelder  that 
we  owe  our  knowledge  with  regard  to  the  laws  which  govern  the  fer- 
mentation of  sugars.  We  have  said  a  little  about  this,  but  it  is  of 
sufficient  importance  to  receive  more  extended  treatment. 

From  the  investigations  of  these  authors,  it  has  been  established 
that  only  those  sugars  are  fermentable,  in  which  the  carbon  atoms 
are  in  multiples  of  three.  The  series  begins  with  glycerol  (C3H6O3), 
the  tetroses  not  being  fermentable,  neither  the  pentoses.  Finally  come 
the  hexoses  which  are  very  fermentable.  These  are  made  up  of  the 
dextroses,  levuloses,  fructoses,  galactoses  and  mannoses.  There  is 
also  a  fermentable  nonose  (C9Hi8O9);  it  is  mannonose  which  is  fer- 
mented by  yeasts  as  easily  as  is  glucose.  After  these  come  the 
bisaccharides  (C^H^On)  and  the  trisaccharides,  melitrioses,  or  raf- 
finoses;  the  melitrioses  have  the  formula  (Ci8H32Oi6).  The  rule 
then  seems  to  be  general.  There  seem,  however,  to  be  certain  excep- 
tions. Thus  it  is  that  the  fermentation  of  glycerose,  always  feeble, 


FERMENTABLE  SUGARS  .  87 

has  been  contested  by  Emmerling.  On  the  other  hand,  Saccharomyces 
thermantitonum  ferments  the  pentoses  (arabinose  and  xylose)  and  it 
seems  to  be  true  with  S.  Ludwigii  according  to  Lindner.  This  author 
found  that  this  yeast  would  ferment  sorbose  and  tagatose  and  that 
Sch.  octosporus  would  ferment  xylose. 

We  have  seen  that  yeasts  are  not  able  to  ferment  polysaccharides, 
but  they  are  broken  up  by  hydrolysis  under  the  action  of  a  special 
enzyme  in  each  case.  It  is  thus  that  starch  is  transformed  by  amylase 
to  maltose,  by  maltase  to  glucose,  saccharose  into  glucose  and  levu- 
lose  by  invertase,  trehalose  into  glucose,  and  lactose  into  glucose 
and  galactose,  etc.  Thus  the  sugars  which  have  a  more  complicated 
structure  are  split  into  C6  sugars  by  enzymes  and  these  in  their  turn 
are  decomposed  into  alcohol  and  carbon  dioxide.  Alcoholic  fermenta- 
tion offers,  then,  one  of  the  best  examples  of  molecular  simplification 
which  the  enzymes  are  able  to  accomplish,  and  suggests  the  important 
role  which  these  bodies  play  in  cellular  life.  We  have  stated  before 
that  the  a-methylglucosides  and  the  /3-methylglucosides  are  ferment- 
able by  certain  yeasts  after  having  been  decomposed  by  maltase  and 
methylglucosases. 

It  has  also  been  established  from  the  work  of  Thierfelder  and 
Fischer  that  the  sugars  with  C6,  Ci2  and  CM  atoms  are  not  ferment- 
able ,to  the  same  degree.  Thus  trehalose  ferments  more  slowly  and 
only  by  certain  yeasts.  Lactose  is  fermented  only  by  a  small  number 
of  yeasts. 

The  same  thing  is  true  with  regard  to  the  hexoses  which  are  also 
not  fermentable  to  the  same  degree.  Among  the  ketohexoses,  for  ex- 
ample, levulose  and  d-fructose  are  alone  fermentable,  and  among 
the  d-glucoses  only  the  d-glucose  (grape  sugar),  d-mannose  and  d- 
galactose  are  fermentable. 

According  to  Thierfelder  and  Fischer,  a  relation  exists  between 
the  fermentability  and  the  structure  of  the  molecule.  In  other  words, 
the  enzymes  have  a  direct  relation  to  the  stereochemic  constitution 
of  the  molecule.  This  relation  has  been  compared  to  that  relation 
which  exists  between  a  key  and  its  lock.  The  aldohexoses  l  suggest  a 
very  important  example  of  this  relation.  Of  the  nine  known  aldo- 
hexoses, there  are  fermentable,  as  we  have  seen,  only  d-glucose,  d- 
mannose  and  d-galactose  with  the  last  possessing  much  less  fermenting 
possibilities  than  the  other  two.  The  chemical  formulae  of  the  aldo- 
hexoses are  the  same  and  differ  only  in  their  molecular  grouping. 
Some  of  these  will  be  given. 

1  The  term  aldohexoses  refers  to  the  hexoses  which  have  an  aldehyde  group 
CHO,  while  the  term  ketohexoses  refers  to  those  which  possass  the  keton  group 
C=0. 


HCO 

HCO 

HCO 

COH 

HCOH 

OHCH 

OHCH 

HCOH 

OHCH 

HCOH 

OHCH 

OHCH 

HCOH 

OHCH 

HCOH 

OHCH 

HCOH 

OHCH 

HCOH 

HCOH 

H2COH 

H2COH 

H2COH 

H2COH 

d-glucose 

1-glucose 

d-mannose 

d-galactose 

88  PHYSIOLOGY  OF  YEASTS 

HCO 

OHCH 

OHCH 

OHCH 

HCOH 

H2COH 

d-talose 

This  indicates  the  differences,  although  very  slight,  in  the  molec- 
ular arrangement  of  the  molecules  which  has  much  effect  on  enzyme 
action.  It  is  thus  that  d-glucose  and  1-glucose  differ  from  one  another 
only  by  the  inverse  position  of  their  molecules.  The  stereochemical 
formula  of  one  represents  the  image  of  the  other  as  seen  in  a  mirror. 
This  structure  is  sufficient,  however,  to  render  d-glucose  fermentable 
and  to  repress  the  fermentability  of  1-glucose.  The  differences  between 
the  grouping  of  d-galactose  and  d-talose  are  very  slight.  So  slight  are 
they  that  d-galactose  is  fermentable  and  d-talose  is  not. 

Among  the  ketohexoses  only  d-fructose  and  levulose  are  ferment- 
able.1 

It  is  proper  to  add  that  the  yeasts  have  a  role  in  the  fermentabil- 
ity of  the  hexoses.  A  true  electivity  has  been  established  for  certain 
sugars.  Dubrunfaut  has  shown  that,  in  a  mixture  of  yeasts,  one  yeast 
will  attack  one  hexose  while  another  will  decompose  another  hexose. 


Formula  of  Alcoholic  Fermentation  and  Secondary  Products 

Alcoholic  fermentation  consists  in  the  transformation  of  sugars 
into  carbon  dioxide  and  ethyl  alcohol.  This  change  is  accompanied 
by  a  liberation  of  heat,  known  for  a  long  time  in  the  fermentation  of 
grape  juice,  and  observed  by  Buchner  in  the  fermentation  induced 
by  yeast  juice.  Bouffard  has  established  a  liberation  of  20  to  23 
calories  for  each  180  grams  of  sugar  destroyed.  A.  Brown  has  ob- 
tained 21.4  calories. 

Gay-Lussac  has  represented  the  alcoholic  fermentation  by  the 
following  simple  equation 

C6H1206  =  2C2H6O  +  2CO2 

This  equation  does  not  take  into  consideration  the  heat  exchange, 
for  it  is  very  difficult  to  express  by  such  a  simple  formula  a  phenome- 
non of  such  complexity.  It  merely  gives  a  general  idea  with  regard 
to  the  change,  but  does  not  take  into  consideration  the  secondary 
products  which  are  formed  during  the  fermentation.  Pasteur  has 
1  Fischer,  E.  and  Thierfeldsr,  H.  Berichte,  27,  1894. 


PROPERTIES  OF  BUCHNER'S  ZYMASE  89 

shown  that  alcohol,  carbon  dioxide,  glycerol,  and  succinic  acid  are 
all  formed  at  the  same  time;  however,  these  products  are  always  in 
small  quantities.  According  to  this  savant,  105.65  grams  of  glucose 
yielded  the  following: 

Ethyl  alcohol 51.11 

Carbon  dioxide    49 . 42 

Succinic  acid    0 . 673 

Glycerol 3.40 

Among  the  products  of  fermentation  may  be  found  such  secondary 
products  as  fatty  acids,  volatile  acids,  ethyl  aldehyde,  acetic  acid, 
higher  alcohols,  ethers,  and  tyrosine  and  leucine.  Some  result  from 
the  decomposition  of  sugars  while  others  are  products  of  excretion 
from  the  cell.  Glycerol  seems  to  belong  to  the  first  category.  The 
volatile  acids  are  probably  connected  with  the  nitrogenous  metabo- 
lism (Duclaux,  Kruis).  According  to  Ehrlich,  it  seems  to  be  the  same 
with  succinic  acid  and  the  higher  alcohols.  It  has  been  established 
by  Ehrlich  that  leucine  and  isoleucine  are  assimilated  by  yeasts 
with  the  fixation  of  a  molecule  of  water;  they  are  decomposed  into 
amyl  and  isoamyl  alcohols  by  the  liberation  of  ammonia  which  serves 
the  metabolism  of  the  yeasts.  The  ethers  result  from  the  action  of 
acids  formed  by  the  action  of  air  or  other  organisms  with  ethyl 
alcohol.  As  to  the  presence  of  aldehydes  established  by  many  investi- 
gators (Roux  and  Linossier,  Duclaux,  Roeser),  they  seem  to  result 
from  the  oxidation  of  alcohol  by  air  or  by  the  action  of  the  yeasts. 
The  observations  of  Trillat  and  Sauton,  including  those  of  Kayser  and 
Demolom,  have  shown  that  wine  yeast;  agitated  in  the  presence  of  air 
caused  a  change  of  a  part  of  the  alcohol  into  aldehydes,  ethyl  and 
acetic.  The  acetic  aldehyde  is  finally  oxidized  to  acetic  acid.  It  then 
seems  as  if  the  yeasts  are  able  to  use  alcohol  which  results  from 
fermentation  and  oxidize  it  to  the  aldehyde. 

Character  and  Properties  of  Buchner's  Zymase 

What  is  the  mechanism  by  which  fermentation  is  accomplished? 
In  1858,  Berthelot  was  the  first  to  demonstrate  that  fermentation 
was  brought  about  by  enzymes  secreted  by  yeasts.  Bernard  toward 
1860  objected  to  this  view.  Pasteur  and  Denys  Cochin  had  tried  to 
isolate  this  enzyme  from  yeast  but  their  efforts  were  in  vain.  Pas- 
teur without  discarding  the  possibility  of  an  enzyme  action  thought 
more  and  more  that  fermentation  was  a  vital  act  of  the  yeast  cell 
itself. 

It  is  known  that  the  future  has  borne  out  Berthelot's  contentions. 
Buchner,  in  1897,  succeeded  in  extracting  the  zymase  from  the  yeast 


90  PHYSIOLOGY  OF  YEASTS 

cell.  This  discovery  demonstrated  that  fermentation  is  able  to  be  pro- 
duced without  the  life  of  the  yeast.  This  made  it  possible  to  abandon 
the  vitalistic  conception  of  alcoholic  fermentation. 

We  have  seen  at  the  beginning  of  this  chapter  how  Buchner  ex- 
tracted his  yeast  juice.  It  is  definitely  settled  today  that  the  juice 
contains  a  zymase  and  that  its  action  is  not  due  to  particles  of  pro- 
toplasm as  certain  investigators  believed  in  the  beginning;  when 
placed  in  contact  with  sugar  it  acts  exactly  like  an  enzyme. 

It  is  not  altered  and  continues  to  act  in  the  presence  of  anti- 
septics (toluol  and  chloroform).  On  the  other  hand  its  efficiency  is 
not  entirely  destroyed  on  filtration  through  a  Chamberland  bougie. 
Finally,  Pay  en  and  Persoon  have  shown  that  yeast  juice  precipitated 
with  alcohol  gives  a  powder  insoluble  in  water  which  possesses  all 
of  the  properties  of  the  juice.  In  order  to  secure  this  powder,  the 
juice  is  precipitated  by  12  times  as  much  alcohol  as  there  is  juice.  A 
mixture  of  800  parts  of  alcohol  and  400  parts  of  ether  may  also  be 
used.  The  precipitate  is  filtered  rapidly,  washed  with  ether,  and 
dried  over  sulfuric  acid  in  a  vacuum. 

The  investigations  of  Albert l  and  those  of  Rapp  have  discovered 
another  method  of  securing  alcoholic  fermentation  away  from  the  liv- 
ing cell.  These  investigators  have  fixed  the  zymase  in  the  cell  by 
acetone  which  killed  the  cell,  without  altering  the  enzymes.  The 
method  consists  of  treating  the  yeast  with  from  10  to  20  times  its 
volume  of  alcohol  ether  or  acetone.  All  of  the  cells  are  killed.  The 
yeast  at  first  is  rid  of  its  water,  is  placed  on  a  filter  paper,  washed 
with  ether,  and  dried  at  45°.  A  white  powder  is  thus  obtained  made 
up  of  dead  cells  which  has "  been  called  zymine  or  durable  yeast 
(Dauerhefe).  The  cells  which  constitute  this  powder  are  endowed 
with  the  fermenting  property  like  living  yeasts  and  when  they  are 
put  into  a  sugar  solution,  they  induce  fermentation  immediately. 
When  this  powder  is  extracted  by  the  method  of  Buchner,  the  juice 
thus  secured  possesses  the  same  action  as  the  living  cells. 

More  recently  Lebedeff  2  has  obtained  a  very  active  zymase  by  the 
maceration  of  the  yeast  in  water.  For  this,  it  is  sufficient  to  mac- 
erate 2.5  to  3  parts  of  water  with  1  part  of  yeast  over  a  period  of 
time.  This  is  finally  filtered  through  filter  paper  and  a  juice  collected 
which  is  very  clear  and  whose  activity  excels  that  secured  by  any  of 
the  other  methods. 

The  quantity  of  zymase  in  living  yeasts  is  variable.     It  is  curious 

1  Albert,  K.      Herstellung   von   Dauerhefe   mittels   Aceton.      Ber.    d.    chem. 
Ges.  31. 

2  Lebedeff,    A.     Extraction   de   la   zymase   par   simple   maceration.      Comp. 
Rend.  Ac.  des  Sciences,  152,  1911. 


PROPERTIES   OF  BUCHNER'S   ZYMASE  91 

to  note  that  the  quantity  of  zymase  in  pressed  yeast  increases  con- 
siderably when  the  yeast  is  kept  at  low  temperatures  and  that  it 
diminishes  in  yeast  during  the  course  of  fermentation.  The  investi- 
gations of  Haydruck  and  Delbriick  have  shown  that  yeasts  cultivated 
in  a  solution  of  sugars  and  mineral  salts  and  removed  at  the  moment 
when  fermentation  is  most  active,  does  not  contain  much  zymase.  If, 
on  the  contrary,  a  yeast  is  taken  from  a  vigorous  fermentation, 
washed,  and  kept  at  a  low  temperature,  the  zymase  content  increases 
rapidly.  (Delbriick,  Buchner,  and  Spitta.)  All  this  seems  to  be  ex- 
plained by  the  fact  that  endotryptase  and  lipase  find  themselves 
affected  by  the  low  temperature  and  do  not  act  on  the  zymase  which 
they  destroy  under  other  conditions. 

In  the  refrigerator  zymase  is  kept  with  difficulty  and  soon  loses 
its  activity  at  the  end  of  one  or  two  days  when  placed  at  ordinary 
temperatures.  At  low  temperatures,  it  is  destroyed  by  degrees. 
This  destruction  was  at  first  explained  by  thinking  that  zymase  was 
easily  oxidized  by  air.  The  investigations  of  Buchner  and  Antoni l 
have,  on  the  contrary,  indicated  that  oxygen  has  no  action  on  zymase 
either  during  periods  of  its  conservation  or  active  fermentation, 
as  they  determined  it.  Its  alteration  arises  from  the  endotryptase 
which  is  associated  with  it  and  perhaps  to  the  lipase  which  one  also 
finds  in  the  juice.  The  work  of  Gromow  and  Grigoriew  indicates  that 
endotryptase  attacks  and  digests  it.  On  the  other  hand,  the  results 
secured  by  Harden,  Buchner,  Wroblewsky  seem  to  indicate  that  lipase 
acts  on  the  coferment.  One  is  able,  however,  to  keep  yeast  juice  in 
all  its  activity  by  drying  it  in  a  vacuum  at  35°  C.  The  juice  is  then 
changed  to  a  yellow  which  may  be  kept  for  a  long  time  unaltered  (10 
or  12  months).  Zymase  also  retains  its  activity  for  a  long  time  if 
preserved  in  a  15  per  cent  solution  of  saccharose,  this  concentration 
acting  on  the  endotryptase. 

The  investigations  of  the  two  English  investigators,  Harden  and 
Young,  have  widened  the  horizon  of  our  knowledge  with  regard  to 
the  constitution  of  zymase.  They  have  shown  that  when  yeast  juice 
is  introduced  into  a  dialyzing  apparatus,  it  may  be  divided  into  two 
parts,  a  non-dialyzable  residue  and  a  liquid  which  does  dialyze.  The 
residue  is  without  fermenting  activity  and  has  been  given  the 
name  of  "inactive  residue."  The  dialyzable  liquid,  which  is  without 
action  on  sugar,  has  been  regarded  as  a  coferment.  Fermentation  is 
only  produced  when  the  two  parts  are  reunited.  The  inactive  resi- 
due may  also  be  regenerated  by  adding  yeast  juice  which  has  been 
submitted  to  boiling,  which  indicates  that  the  coferment  is  able  to  re- 

1  Buchner,  E.  and  Antoni,  W.  Weitere  Versuche  iiber  die  zellfreie  Garung. 
Zeit.  phys.  Chem.  44,  1905. 


92  PHYSIOLOGY  OF  YEASTS 

sist  boiling  temperatures.  The  investigations  of  Buchner,1  Hoffman, 
Duchacek,1  Klatte,2  Hoehn  3  and  Resenbeck  have  confirmed  the  exist- 
ence of  a  coferment.  In  adding  this  yeast  juice  to  a  double  vol- 
ume of  boiled  juice  these  investigators  have  noticed  an  increase  in  the 
activity,  almost  proportional  to  the  amount  of  boiled  juice  added. 
On  the  other  hand  the  addition  of  inactive  boiled  juice  to  the  yeast 
juice,  which  had  become  inactive,  restored  the  fermenting  activity. 
This  demonstrated  that  in  old  juice  there  is  active  zymase  but  that 
it  lacks  a  coferment.  This  coferment  seems,  then,  to  disappear  during 
fermentation  before  the  zymase.  Gromow  and  Grigoriew  have  also 
reported  that  if  fresh  zymase  is  added  to  a  zymase  which  is  becoming 
inactive,  more  fermentation  is  secured  than  if  the  fresh  zymase  was 
used  alone.  The  old  zymase  has  ceased  to  act  on  acccount  of  the  al- 
teration of  its  coferment  and  the  addition  of  the  fresh  zymase  regen- 
erates it. 

From  all  of  this  has  been  established  that  zymase  may  result  from 
a  mixture  of  two  substances:  a  dialyzable  body  which  resists  boiling, 
the  coferment,  and  a  substance  little  or  not  dialyzable,  which  does 
not  resist  boiling.  It  is  only  by  the  union  of  the  two  bodies  that 
fermentation  takes  place. 

The  investigations  of  Wroblewsky,  Buchner,  Harden  and  Young 
have  permitted  some  explanations  of  the  nature  of  the  coferment. 
These  authors  have  stated  that  the  addition  of  phosphate  salts  of 
sodium  or  potassium  will,  like  the  ferment,  produce  an  accelera- 
tion in  fermentation.  The  addition  of  serum  or  lecithin  produces 
the  same  effect.  The  coferment  loses  its  activity  by  heating  for  4 
hours  in  water  at  130°  C.  It  is  not  attacked  by  trypsin  but  is  destroyed 
by  the  lipase  which  exists  in  the  yeast  juice.  The  action  of  this  lip- 
ase  in  the  yeast  juice  seems  then  to  be,  with  the  endotryptase,  the 
principal  cause  of  the  rapid  loss  of  zymase  activity.  The  lipase 
acts  on  the  coferment  and  the  endotryptase  on  the  zymase.  The 
coferment  is  present  in  lesser  quantities  than  the  zymase.  It  is  able 
to  be  kept  in  sugar.  This  represses  the  action  of  proteolytic  enzymes 
and  perhaps  the  lipase.  In  this  way,  the  action  of  these  strong  sugar 
solutions  may  be  explained.  Later  on  all  these  facts  will  be  of  much 
interest  in  the  discussion  of  the  mechanism  of  the  alcoholic  fermen- 
tation. 

1  Buchner,    E.    and   Duchacek,    E.     Ueber   fraktionierte    Fallung   des  Hefe- 
pressaftes.    Biochem.  Zeitschr.  15,  1909. 

2  Buchner,  E.  and  Klatte,  F.    Ueber  das  Koenzym  in  Hefepressaft.    Biochem. 
Zeitschr.  19,  1909. 

3  Buchner,  E.  and  Hoehn,  H.     Ueber  das  Spiel  der  Enzyme  in  Hefepressaft. 
Biochem.  Zeitschr.  19,  1909. 


PROPERTIES  OF  BUCHNER'S  ZYMASE  93 

The  investigations  of  Buchner  and  his  collaborators  have  revealed 
the  presence  of  a  substance  in  boiled  juice  which  protected  the  zymase 
from  the  action  of  the  endotryptase.  This  has  received  the  name  an- 
tiprotease.  This  enzyme  protects  gelatin  and  milk,  also,  from  the 
action  of  the  endotryptase  in  yeast  juice.  It  prevents  the  liquefac- 
tion of  milk  and  the  peptonization  of  gelatin. 

The  existence  of  this  antiprotease  permits  an  explanation  of  how 
the  fermenting  action  is  preserved  for  many  days  when  boiled  juice  is 
added  to  fresh  juice;  otherwise  it  would  disappear  rapidly.  In  the 
yeast  juice,  without  the  addition  of  the  boiled  juice,  an  almost  complete 
disappearance  of  protein  substances  was  noticed  after  7  days.  When 
boiled  juice  is  added  no  precipitation  of  protein  occurs.  The  addi- 
tion of  the  boiled  juice  seems,  then,  to  protect  the  fresh  juice  for  a 
period  of  time  against  the  proteolytic  action  of  the  endotryptase. 
The  experiments  of  Buchner  indicate  that  it  will  protect  also  from  the 
action  of  pepsin  and  trypsin.  This  proves,  then,  that  zymase  is  a 
protein  substance.  Careful  experiments  have  indicated  that  it  is 
possible  to  destroy  the  coferment  of  boiled  juice  without  destroying 
the  antiprotease.  This  may  be  accomplished  by  heating  it  for  several 
hours.  The  juice  thus  treated  still  exerts  a  protective  action  towards 
gelatin  and  yeast  juice.  It  contains,  then,  an  antiprotease.  On  the 
contrary,  this  juice  is  not  capable  of  regenerating  the  preserved  yeast 
juice  because  it  contains  no  coenzyme.  The  antiprotease  is,  then,  dis- 
tinct from  the  coenzyme.  It  does,  however,  have  some  likenesses 
to  the  latter;  it  is  destroyed  by  lipase  and  seems  to  be  a  saponifiable 
ether  of  phosphoric  acid.  The  antiprotease  seems  to  play  an  impor- 
tant role  in  the  life  of  the  yeast  and  regulates  its  digestive  functions 
(especially  autolysis) . 

Zymase  acts  best  in  an  alkalin  medium.  The  addition  of  sodium 
carbonate  and  phosphate  exerts  a  favorable  action.  It  is  destroyed  by 
heating  to  55°;  in  the  dry  condition,  if  desiccation  has  been  carried 
out  in  vacuo  at  40°,  it  is  able  to  resist  140°.  Temperature  exerts  a 
decided  influence  on  the  activity  of  zymase  because  the  action  of  endo- 
tryptase and  lipase  on  it  is  much  altered  with  the  temperature.  That 
temperature  at  which  zymase  exerts  the  greatest  fermenting  action  is 
about  14°.  The  optimum  temperature  seems  to  be  higher;  but  endo- 
tryptase will  attack  zymase  when  the  temperature  is  even  higher. 

Concentration  plays  a  r61e  in  the  activity  of  zymase.  Fermenta- 
tion increases  with  the  concentration  of  the  sugar;  this  explains  the 
relation  of  concentrated  sugar  solutions  (15  per  cent)  to  the  suppres- 
sion of  endotryptic  action.  The  optimum  concentration  of  sugar 
seems  to  be  about  25  per  cent. 

The  yeast  juice  contains,  as  we  have  said,  hydrolytic  enzymes  for 


94  PHYSIOLOGY  OF  YEASTS 

carbohydrates  (maltase,  invertase).  The  hexoses  are  fermented  im- 
mediately and  the  fermentable  Ci2  and  Cis  sugars  are  transformed  to 
hexoses  by  means  of  these  enzymes.  It  contains  also  a  glycogenase 
which  allows  it  to  induce  the  fermentation  of  glycogen  upon  which  the 
living  yeast  has  no  action;  the  glycogenase  is  diffusible  while  the 
glycogen  is  not. 

On  the  contrary,  the  ordinary  juice  does  not  act  on  lactose,  but 
Buchner  and  Meisenheimer  have  been  able  to  isolate  from  lactose- 
fermenting  yeasts  a  lactase. 

One  is  easily  able  to  secure,  by  mixing  some  of  Buchner's  yeast 
juice  with  a  solution  of  glucose,  a  fermentation  which  will  start  at 
the  end  of  six  minutes:  20  c.c.  of  yeast  juice,  with  8  grams  of  saccha- 
rose in  the  presence  of  0.2  c.c.  of  toluol  will  yield  after  96  hours 
from  0.7  to  1.87  grams  of  carbon  dioxide.  If  these  results  are  com- 
pared with  those  obtained  with  living  yeast,  the  fermenting  power  of 
the  juice  seems  feeble,  for  1  gram  of  living  yeast  will  produce  from 
an  8  per  cent  solution  of  sugar  at  40°  in  about  6  hours,  1.5  grams  of 
CO2.  Zymase  is  not  extracted  in  a  pure  state  and  it  must  be  admitted 
that  it  makes  up  only  a  feeble  part  of  the  juice.  However,  in  the 
fermentation  with  the  living  yeast,  new  zymase  is  formed  constantly. 

Among  the  secondary  products,  glycerol  (3  to  8  per  cent),  traces 
of  acetic  acid  and  amyl  alcohol,  have  been  noticed.  Lactic  acid  is 
often  found  but  it  may  disappear  and  may  be  transitory.  Further  on, 
we  shall  see  the  significance  of  the  formation  of  this  lactic  acid. 


Mode  of  Action  of  Zymase 

For  a  long  time  the  following  formula  has  expressed  alcoholic  fer- 
mentation 

C6Hi206  =  2(C2H5OH)  +  2CO2. 

This  equation  has  only  general  value.  Among  the  two  products  ex- 
pressed, there  are  many  intermediate  products.  The  determination  of 
these  has  demanded  the  sagacity  of  the  biochemists.  It  might  be 
advisable  to  give  some  of  the  principal  results  which  have  been  ob- 
tained. 

After  the  work  on  oxidases  and  hydrogenases  of  yeasts  Griiss  l 
has  put  out  a  theory  to  explain  the  mechanism  of  alcoholic  fermen- 
tation which  is  very  interesting.  This  author  regards  glycogen  as  an 
intermediate  product  between  fermentation  and  respiration.  The 
polysaccharides  will  be  decomposed  into  glucose  by  means  of  the 
hydrolytic  enzymes  (sucrase  and  maltase) ;  this  will  be  split  into  two 
1  Gruss,  J.  Zeitschr.  f.  ges.  Brau.  27,  1904. 


MODE  OF  ACTION   OF  ZYMASE  95 

CH2OH 

I 
groups  by  the  action  of  the  zymase  CH  OH.    These  groups  combine 

COH 

with  the  protoplasm.  The  protoplasm  will  secrete  glycogen  which 
will  finally  be  hydrolyzed  into  glucose  by  the  glycogenase.  Two  con- 
ditions are  then  possible:  when  the  yeast  is  in  the  presence  of  air  and 
when  it  does  not  have  air  at  its  disposition. 

In  the  first  case,  under  the  influence  of  oxygenase,  put  in  evidence 
by  Griiss,  the  molecules  of  glucose  are  decomposed  into  the  above 
grouping  which  are  oxidized  by  the  oxygen  liberated  by  oxygenase 
and  thus  changed  into  carbon  dioxide  and  water.  The  reaction  may 
be  expressed  by  the  following  equation: 

CH2OH-CHOH-COH  +  6O  =  3C02  +  3H20. 

This  is  ordinary  aerobic  respiration. 

In  the  second  case,  the  enzyme  which  we  have  come  to  know  under 
the  name  of  hydrogenase  acts  on  the  products  of  decomposition  from 
glucose.  Owing  to  the  intervention  of  water,  CO2  will  be  formed  in 
a  first  phase  with  a  liberation  of  hydrogen.  In  the  second  phase  these 
hydrogen  atoms  will  be  used  to  unite  with  the  rest  of  the  glucose 
molecule. 


CH2OH-CHOH-COH  +  3H2O  =  3C02  +  12H. 

.rnase 

Second    2(CH2OH-CHOH-COH)  +  12H  =  3C2H5OH  +  3H2O. 

JL  nase 

This  theory  of  Griiss,  in  spite  of  its  complexity,  has  the  advan- 
tage of  explaining  the  role  of  oxygenase  and  hydrogenase. 

Another  theory  has  been  expounded  by  Wohl.  It  has  been  stated 
that  small  quantities  of  lactic  acid  are  often  found  among  the  prod- 
ucts of  alcoholic  fermentation.  Some,  especially  Buchner  and  Mei- 
senheimer,  have  regarded  this  acid  as  intermediary  in  the  mechanism 
of  the  decomposition  of  glucose  into  alcohol  and  carbon  dioxide.  This 
may  be  regarded  as  taking  place  in  two  phases.  In  the  first  phase, 
the  zymase  transforms  the  glucose  into  lactic  acid,  and  in  the  second, 
another  enzyme,  the  lactacidase,  decomposes  the  lactic  acid  into  al- 
cohol and  carbon  dioxide.  However,  Buchner  and  Meisenheimer  have 
not  succeeded  in  transforming  lactic  acid  into  alcohol  and  C02  by  yeast. 
This  theory  has  been  attacked  by  Slator,  and  Buchner  himself  has 
given  it  up.  It  may  be  regarded  as  of  classic  interest  only. 

One  is  not  able  to  give  up  entirely  the  idea  that  a  large  molecule 
like  glucose  is  not  able  to  be  split  immediately  into  small  fragments. 


96  PHYSIOLOGY  OF  YEASTS 

It  ought  to  have  intermediary  products.  Quite  recently,  Wohl  and 
also  Buchner,1  Boyen-Jensen,  and  Fernbach  2  have  admitted  that  this 
intermediate  product  may  be  dioxyacetone  with  the  formula  CH2OH- 
CO-CH2OH. 

This  compound,  under  certain  rare  conditions,  is  able  to  yield  lac- 
tic acid.  But,  more  often,  this  dioxyacetone  will  give  alcohol  and 
carbon  dioxide  directly.  Alcoholic  fermentation  then  acts  in  two 
phases:  in  the  first,  the  glucose  is  transformed  into  dioxyacetone,  and 
in  the  second  phase,  a  dioxyacetonase  will  change  the  dioxyacetone 
into  alcohol  and  carbon  dioxide.  Zymase  will  then  in  reality  be  made 
up  of  two  enzymes  acting  successively. 

Buchner  and  Meisenheimer  have  been  able  to  obtain  fermentation 
of  dioxyacetone  (2  per  cent  solution)  by  yeast  juice.  Lebedeff3  quite 
recently  obtained  good  results  when  making  yeast  juice  ferment  a 
5  per  cent  solution  of  dioxyacetone.  All  of  these  facts  are  very  in- 
teresting, but  it  still  remains  true  that  the  demonstration  of  the 
formation  of  dioxyacetone  during  fermentation  has  not  been  accom- 
plished. Its  existence  is,  then,  purely  theoretical.  We  have  cited  the 
work  of  Harden  4  and  Young,  who  have  demonstrated  that  zymase  is 
composed  of  two  elements,  one  a  dialyzable  and  thermostabile,  the 
other  not  dialyzable  and  destroyed  at  100°  C.  This  last  does  not 
possess  any  fermenting  function.  If  one  adds  to  it  the  coenzyme,  fer- 
mentation will  result  immediately. 

But  another  agent  has  been  found  which  will  activate  yeast  juice 
which  has  no  or  little  activity;  it  is  the  phosphates  of  either  sodium 
or  potassium.  If  a  little  soluble  phosphate  is  added  to  yeast  juice 
a  brisk  liberation  of  CO2  results.  This  exists  for  a  tune  proportional 
to  the  quantity  of  phosphate  added,  then  it  slows  up  and  fermenta- 
tion goes  on  as  before.  If  phosphate  is  added  again  the  phenomenon 
is  repeated.  One  is  thus  able  to  reproduce  it  a  number  of  times.  The 
addition  of  phosphates  then  has  the  same  effect  as  the  addition  of 
a  coenzyme  or  the  boiled  inactive  juice  alone. 

Such  are  the  facts  which  the  investigations  of  Harden  and  Young 
and  Lebedeff  have  established.  An  ingenious  conception  of  the 
mechanism  of  alcoholic  fermentation  has  thus  been  formed. 

1  Buchner,  E.     La  fermentation  alcoolique  des  sucres.     Rev.  g.  des  Sciences, 
21,  1910. 

2  Fernbach,  A.    Stir  la  degradation  des  hydrates  de  carbonne.     C.  R.  Acad. 
des  Sciences,  150,  1910. 

3  Lebedeff,    A.     Sur   le   me'canisme   de   la   fermentation   alcoolique.     Comp. 
Rendu   Acad.  Sciences,    153,    1911;     Ueber   Hexosphosphorsauerester.    Biochem. 
Zeitschr.  27,  1910. 

4  Harden,    A.      Recherches   recentes   sur   la   fermentation   alcoolique.      Ann. 
de  la  Brasserie  et  de  la  Distillerie,  14th  Year, -1911. 


MODE  OF  ACTION  OF  ZYMASE  97 

The  phosphate  added  enters  into  a  combination  with  the  glucose. 
What  demonstrates  this  is  the  fact  that  the  phosphate  is  not  able  to 
be  obtained  again  by  the  usual  magnesium  salt.  Harden  and  Young 
have  thought  that  the  phosphate  united  to  two  half  molecules  of  the 
hexose.  The  remaining  half  molecules,  with  three  atoms  of  carbon, 
gave  alcohol  and  carbon  dioxide.  The  combination  of  hexose-phos- 
phate  is  transformed  by  an  enzyme  hexose-phosphatase  which  re-forms 
a  new  combination  with  two  remnants  of  the  hexoses.  The  phos- 
phate, then,  follows  a  cycle  and  it  is  the  quickness  with  which  the 
phosphate  traverses  this  cycle  that  determines  the  speed  of  fermenta- 
tion. 

Lebedeff  has  produced  an  important  contribution  in  relation  to 
this  conception.  He  has  been  able  to  prepare  the  osazones,  phenyl- 
and  bromophenylhydrazones  of  this  hexose-phosphate.  He  has  iso- 
lated and  analyzed  the  calcium  salt.  The  following  formula  is  given 
to  this  body  C6HioO4(R2PO4)2,  resulting  from  the  condensation  of  two 
molecules  of  C3H5O2I^PO4. 

Young  developed  the  following  formula  with  either  an  aldehyde 
or  ketone  group  free  to  form  hydrazones: 

CHO 

CH-P04H2 
(CH-OH)3 
CH-P04H2 

According  to  Lebedeff  alcoholic  fermentation  takes  place  accord- 
ing to  the  following  equations: 

1.  C6Hi2O6  =  2  (C3H603). 

2.  2  (C3H6O3)  +  2  RHPO4  =  2  (C3H5O2RPO4)  +  2  H2O. 

3.  2  (C3H5O2RPO4)  =  C6H10O4(RP04)2. 

4.  C6H1004(RP04)2  +  H20  =  C2H5OH  +  CO2  +  C3H5O2  +  2  (RHP04) 

or  even 

5.  C6H1004(RP04)2  +  2  H20  =  2  (C2H5OH)  +  2  C02  +  2  (RHPO4). 

This  scheme  involves  the  following; 

Decomposition  of  the  hexose  into  2  molecules  of  triose  (dioxy- 
acetone). 

Union  of  one  molecule  of  the  triose  with  1  molecule  of  phosphate; 
a  phosphoric  ether  results. 

Condensation  of  two  molecules  of  this  phosphoric  ether  with  one 
molecule  of  hexose-phosphate. 

Decomposition  of  this  hexose-phosphate  into  phosphate,  alcohol 
and  carbon  dioxide. 


98  PHYSIOLOGY  OF  YEASTS 

This  question,  as  we  have  seen,  is  extremely  complex;  the  question 
is  being  studied  but  instead  of  becoming  simpler  becomes  more  com- 
plicated. 

Lebedew  l  has  later  restated  his  idea  of  the  mechanism  of  alco- 
holic fermentation  as  follows: 

4  C6Hi2O6  =  8  C3H6O3. 

Glyceraldehyde 
4  C3H603  -  4  H2  =  4  C3H403. 
4  C3H403  =  4  C2H4O  +  4  CO2. 
4  C2H40  +  4  H2  =  4  C2H5-OH. 

Dioxyacetone 

4  C3H6O3  +  4  RHPO4  =  4  G3H5O2RPO4  +  4  H20. 
4  C3H5O2RPO4  =  2  C6H10O4(RPO4)2. 
2  C6H1004(RP04)2  +  4  H20  =  2  C6H12O6  +  4  RHPO4. 
2  C6H1206  =  4  C3H603,  etc. 

Neuberg  2  has  studied  the  possibilities  of  intermediate  compounds 
in  alcoholic  fermentation.  He  believes  that  the  CO2  which  appears 
in  alcoholic  fermentation  is  split  off  from  (CH3CO.COOH)  pyruvic 
acid.  He  found  that  it  took  from  2-8  seconds  for  CO2  to  be  formed  in 
yeast  maceration  +  CH3CO.COOH  while  it  took  from  2-3  hours  for 
it  to  be  formed  from  yeast  +  glucose.  Neuberg  and  his  coworkers 
also  used  oxalacetic  acid  and  found  that  yeast  would  change  this  to 
two  molecules  of  CO2  and  one  molecule  of  CH3CHO.  Hydroxypyruvic 
acid  yielded  CO2  and  glycoaldehyde,  a  ketobutyric  acid  yielded  pro- 
pionaldehyde  and  C02  along  with  some  propyl  alcohol.  They  find 
that  the  cleavage  of  pyruvic  acid  takes  place  2000  times  more  rapidly 
than  the  complete  fermentation  of  sugar.  Neuberg  and  Kut  then 
argue  that  two  processes  are  involved  in  alcoholic  fermentation. 
First,  one  class  of  enzymes  hydrolyzes  the  C6  molecule  into  C3  chains. 
Secondly,  another  class  of  enzymes  (carboxylase)  breaks  the  C3  chains 
up  into  C2  and  Ci  compounds.  In  further  researches  Neuberg  has 
shown  that  yeasts  possess  an  enzyme  which  decomposes  pyruvic  acid 
into  acetaldehyde  and  C02.  Acetaldehyde  is  readily  reduced  during 
fermentation  to  ethyl  alcohol.  From  this  it  seems  probable  that 
aldehyde  is  an  intermediate  compound  in  fermentation.  Neuberg 
and  Reinfurth  showed  that  if  fermentation  was  carried  out  in  the 
presence  of  NasSOs  considerable  amounts  of  aldehyde  could  be  ob- 

1  Lebedew,  A.  and  Griaznoff,  N.      Ueber  den  Mechanismus  der  alkoholischen 
Garung.     II.  Chem.  Berichte,  45  (1912),  3256-3272. 

2  See   bibliographical  index   for   complete   list   of   Neuberg's  publications  on 
sugar-free  fermentations. 


PASTEUR'S  THEORY  99 

tained.  The  reactions  illustrating  the  cleavage  of  glucose  according 
to  Neuberg's  theory  may  be  written  as  follows  according  to  Euler 
and  Lindner: 

C6Hi2O6  -  2  H20  =  C6H804  (Metbylglyoxal-aldol) 
C6H804  =  2  CH2:C(OH).COH  or  2  CH3.CO.COH  (Methyl- 

glyoxal) 
CH2 :  C(OH).COH  +  H2O  H2     CH2OH-CHOH-CH2OH 

+       =  Glycerol 

CH2:C(OH).COH  O      CH2:  C(OH).COOH 

Lactic  acid 

CH3.CO.COOH  =  CO2  +  CHa.COH  (Acetaldehyde) 
CH3.CO.COH        O      CH3.CO.COOH  (Lactic  acid) 

+  1  - 
CH3.COH  H2     CH3.CH2OH  (Ethyl  alcohol) 

Neuberg l  has  produced  more  evidence  to  support  his  aldehyde 
theory  of  fermentation.  By  adding  sodium  sulfite,  aldehyde  and  glyc- 
erol  were  the  chief  products. 

Lob 2  has  given  the  chemistry  of  alcoholic  fermentation  comprehen- 
sive study.  He  has  produced  much  evidence  on  the  fact  that  aldehyde 
is  the  important  intermediate  compound.  He  argues  that  the  sugars 
tend  to  cleave  into  the  same  substances  from  which  they  may  be  built 
up.  That  aldehydes  are  intermediate  in  alcoholic  fermentation  has  been 
stated  by  many,  but  few  have  gone  far  enough  to  produce  either 
plausible  evidence  or  experimental  data  to  support  their  claims. 

Kusserow  3  proposed  that  glucose  was  first  reduced  to  sorbitol  and 
this  fermented. 

General  Theories  of  Alcoholic  Fermentation 

Alcoholic  fermentation  seems  to  have  for  its  purpose  the  libera- 
tion of  the  energy  necessary  in  the  life  of  the  yeast  when  it  finds 
itself  deprived  of  air  under  conditions  in  which  respiration  is  not  pos- 
sible. This  theory  has  not  been  accepted  by  all  and  it  might  be 
well  to  mention  some  of  the  different  theories  which  have  been  put 
forth  to  explain  this  phenomenon. 

Pasteur's  Theory 

Pasteur  was  the  first  to  think  that  yeasts,  when  growing  away 
from  air,  might  seek  the  oxygen,  which  they  needed,  in  the  com- 

1  Neuberg,  C.,  and  Reinfurth,  E.  Natural  and  forced  glycerol  formation  in 
alcoholic    fermentation.      Biochem.    Zeit.    92,    234-66,    1918.     Chem.    Absts.    13 
(1919),  2046. 

2  Lob,  W.    See  bibliographical  index. 

3  Kusserow,  R.     Eine  neue  Theorie  der  alkoholischen  Garung.     Cent.  Bakt. 
Abt.  II.,  26  (1910),  184-187. 


100  PHYSIOLOGY  OF  YEASTS 

pounds  which  were  accessible  to  them.  By  decomposing  these,  they 
are  able  to  get  this  oxygen.  Alcoholic  fermentation  would  then  be  a 
method  for  resisting  suffocation.  The  yeast,  not  finding  oxygen  avail- 
able and  not  being  able  to  live  without  this  element,  will  be  obliged 
to  take  it  from  some  of  its  combinations  in  sugar  which  they  find  in 
the  medium. 

The  same  thing  takes  place  in  other  living  beings.  The  yeasts 
possess,  then,  the  property  in  common  with  other  organisms,  but  they 
are  better  adapted  than  the  others  to  produce  fermentation  and  thus 
resist  suffocation. 

In  short,  states  Pasteur,  nearly  all  known  beings  without  excep- 
tion are  only  able  to  respire  and  sustain  themselves  by  assimilating 
free  gaseous  oxgyen;  there  is  a  class,  however,  in  which  respiration 
will  be  quite  active  in  order  that  they  may  live  away  from  air  by  tak- 
ing their  oxygen  from  certain  combinations,  from  which  results  a  slow 
and  progressive  decomposition.  This  last  class  of  organized  beings 
will  be  made  up  of  the  ferments  entirely  similar  to  the  beings  of  the 
first  class,  living  with  them,  assimilating  carbon,  nitrogen  and  phos- 
phates, having  like  them  a  need  for  oxygen,  but  differing  from  them  in 
that  they  are  able  to  respire  with  oxygen  taken  from  compounds, 
when  free  oxygen  is  not  available. 

Pasteur's  theory  has  precipitated  numerous  objections  among 
which  is  this,  that  the  classic  formula  of  Gay-Lussac  accepted  by 
Pasteur  does  not  leave  a  place  for  the  setting  free  of  oxygen. 

This  theory  of  Pasteur's  has  been  modified.  Alcoholic  fermenta- 
tion has  always  been  considered  as  a  phenomenon  of  resistance  to 
suffocation  and  that  it  has  for  its  purpose  the  securing  of  energy 
for  the  life  of  the  yeast.  The  fermentation  will  be,  then,  from  the 
viewpoint  of  energy,  the  equivalent  of  respiration.  The  yeast,  during 
fermentation,  continues  to  develop,  making  new  tissue  and  retaining 
its  usual  functions.  The  yeast  carries  on  a  slow  deliberate  decomposi- 
tion in  quest  of  its  energy  and  it  is  alcoholic  fermentation  which  fur- 
nishes it.  The  yeasts  are  constructed  to  live  in  contact  with  or  away 
from  air.  In  the  first  case,  they  burn  carbohydrates;  in  the  other, 
they  cause  a  breaking  up  or  shifting  of  parts  of  a  complex  molecule. 
But  the  sources  of  cellular  life  are  the  same  in  each  case. 

Theory  of  Wortmann  and  Delbriick 

Wortmann  and  later  Delbriick  have  regarded  alcoholic  fermenta- 
tion as  a  phenomenon  comparable  to  the  secretion  of  a  toxin.  In 
this  case  the  alcohol  serves  the  role  of  a  poison  with  which  the  yeast 
is  able  to  compete  with  other  organisms  with  which  it  comes  in  con- 


THEORY   OF  WORTMANN   AND   DELBRttCK         101 

tact.  The  yeasts  are  able  to  withstand  strong  doses  of  alcohol,  suf- 
ficient to  kill  other  organisms.  They  are  able  to  live  in  a  medium 
which  contains  from  10  to  18  per  cent  of  alcohol  while  other  organisms 
are  killed  by  from  4  to  10  per  cent.  Generally  alcohol  is  not  assimi- 
lated by  yeasts  and  it  is  then  useless  to  them. 

The  first  yeasts,  which  were  without  doubt  the  wild  yeasts,  lived 
like  other  fungi  in  contact  with  air.  But  having  taken  on  the  ability 
of  living  in  decaying  fruits  and  in  the  mucous  secretions  of  trees  in 
order  to  secure  sugar,  they  have  competed  with  other  organisms 
which  lived  under  the  same  conditions.  In  this  struggle  for  life, 
the  yeast  has  been  victorious  against  its  adversaries  and  has  sur- 
vived, thanks  to  the  fermenting  function  which  constitutes  a  means 
of  preservation  for  it.  Yeasts  are  then  adapted  to  live  after  a  special 
manner  away  from  air,  secreting  a  large  amount  of  alcohol  which  acts 
as  a  toxin. 

The  culture  of  yeasts  by  man  has  finally  adapted  them  to  anaer- 
obic life  and  caused  them  to  secrete  increasingly  large  amounts  of 
alcohol.  According  to  this,  the  primitive  yeasts  were  aerobic  and 
slowly  adapted  themselves  to  anaerobic  life. 

Experience  has  demonstrated  that  if  a  new  wine  is  exposed  to  the 
air,  a  vigorous  growth  of  fungi  takes  place  on  the  surface,  consisting 
of  Botrytis,  Penicillium,  Dematium,  bacteria,  and  wild  and  cultivated 
yeasts.  These  organisms  live  along  together  for  a  time  but  as  the 
yeasts  produce  alcohol,  the  medium  becomes  unfavorable  for  some  of 
these  fungi  and  the  bacteria  drop  out.  The  wild  yeasts  are  able  to 
withstand  these  quantities  of  alcohol  for  a  time  but  they  in  turn  are 
killed  as  the  concentration  of  alcohol  increases.  Finally,  by  means 
of  their  adaptation  to  alcohol,  the  cultivated  yeasts  are  able  to  be 
triumphant  over  all  of  the  various  fungi  which  were  present  at  first. 

The  phenomenon  of  alcoholic  fermentation  permits  the  yeasts  to 
resist  suffocation.  By  means  of  it  they  form  alcohol  and  C02  and  thus 
secure  the  heat  which  is  necessary  for  their  maintenance.  Wort- 
mann  and  Delbriick  join  the  theory  of  Pasteur  but  their  hypothesis 
has  the  merit  of  explaining  the  origin  of  alcoholic  fermentation. 

The  theory  of  toxin  formation  has  raised  certain  objectors  who 
claim  that  the  toxin  is  not  generally  secreted  in  sufficient  quantity  to 
injure  the  organisms  which  make  it,  while  the  yeast  produces  such 
quantities  of  alcohol  that  it  is  finally  killed. 

Theory  which  Makes  Fermentation  a  Pnase  of  Respiration 

Another  theory,  which  seems  to  depend  upon  Pasteur's,  is  that 
making  fermentation  a  phase  of  respiration.  It  rests  upon  the  fre- 


102  PHYSIOLOGY  OF  YEASTS 

quency  of  the  formation  of  alcohol  in  living  tissue.  We  have  seen 
that  alcoholic  fermentation  is  not  a  phenomenon  exclusive  to  the  yeast 
but  is  met  among  most  fungi  and  also  in  tissues  containing  sugar. 
Alcohol  seems  to  be  rather  frequently  produced  in  cells.  Berthelot, 
Devaux,  and  Maze  have  found  alcohol  in  a  great  number  of  plants 
placed  under  normal  conditions.  Bechamp  has  isolated  alcohol  from 
the  brains  of  sheep. 

Therefore,  from  these  contributions,  certain  authors  think  (Wort- 
mann,  Polszeniuz,  Goldewski,  Maze l  and  Duclaux,  Pfeffer,  Palla- 
dinn,2  Stoklasa,  etc.)  that  zymase  exists  in  all  organisms  and  func- 
tions in  the  usual  manner.  For  these  investigators,  alcohol  is  always 
an  intermediate  product  in  respiration  of  plants  and  animals.  Res- 
piration, according  to  this,  is  made  up  of  two  phases.  In  the  first,  or. 
intramolecular  respiration,  there  is  no  need  of  concourse  with  oxygen; 
the  sugar  is  decomposed  into  alcohol  and  carbon  dioxide  by  zymase. 
This  is  intramolecular  respiration  or  alcoholic  fermentation.  In  the 
second  phase,  the  alcohol,  thus  formed,  will  be  changed  in  the  pres- 
ence of  air  by  means  of  oxidase  into  carbon  dioxide  and  water. 
This  is  respiration,  properly  speaking,  or  external  respiration.  In  the 
absence  of  air,  the  phenomenon  will  stop  with  the  formation  of  al- 
cohol. 

Thus,  to  express  this  theory  with  formulae,  the  zymase  would 
accomplish  the  following: 

C6H12O6  =  2  C2H6O  +  2  CO2. 

The  second  phase  which  takes  place  in  contact  with  air  is  as  follows: 
C2H6O  +  3  02  =  2  C02  +  3  H2O. 

In  case  that  an  organism,  or  yeast,  finds  itself  away  from  air  the 
change  will  stop  at  the  first  stage.  As  it  goes  on  with  much  inten- 
sity, it  will  give  sufficient  energy  for  those  organisms  which  are  able 
to  live  without  air  like  the  yeasts.  One  finds  here,  then,  a  point  of 
departure  from  Pasteur's  theory. 

Other  authors  go  farther.  They  admit  that  alcohol  is  not  only 
a  product  of  respiration,  but  also  term  it  a  more  simple  assimilation 
of  carbon.  The  hydrocarbon  elements  may  then  be  transformed  into 
alcohol  before  being  assimilated.  According  to  this,  the  hexoses  which 
are  formed  from  the  polysaccharides  by  the  various  enzymes  will  be 
changed  into  alcohol  by  the  zymase.  If  the  phenomenon  takes  place 
in  contact  with  air,  a  part  of  the  alcohol  is  oxidized  by  the  oxidase, 

1  Maze,  P.    La  respiration  des  plantes  vertes;  theorie  biochimique  et  the"orie 
de  la  zymase.    Rev.  g.  des  sciences,  1906.     No.  17. 

2  Palladinn,  W.    Sur  la  respiration  des  plantes.     Biochem.  Zeitschr.  18,  1909. 


THEORY   OF  WORTMANN  AND  DELBRUCK         103 

the  enzyme  of  respiration,  the  other  is  utilized  for  the  maintenance 
of  the  organism  and  the  construction  of  new  tissue.  Zymase  is  then 
an  enzyme  of  digestion  the  same  as  amylase,  maltase,  etc.  If,  on  the 
contrary,  the  phenomenon  takes  place  away  from  air  some  of  the  al- 
cohol will  remain  unutilized,  as  is  the  case  in  the  alcoholic  fermentation 
by  yeasts. 

According  to  Duclaux,  the  term  alcohol  is  not  the  simplest;  by 
means  of  the  oxidases  according  to  him  it  may  be  changed  into  the 
aldehyde.  Many  have  noticed  the  presence  of  alcohol  in  the  product 
of  fermentation.  It  is  known  that  aldehydes  are  generally  considered 
as  the  first  step  in  the  combination  of  H^O  and  CO2  in  the  green  plant. 
The  yeast  acts,  then,  exactly  as  a  higher  plant.  It  assimilates  hydro- 
carbon substances  and  forms  formaldehyde  as  in  the  chlorophyll 
synthesis. 

This  theory  is  supported  by  a  number  of  facts  which  are  inter- 
esting enough  to  cite  at  this  time.  Thus,  Laborde  has  called  attention 
to  a  mold,  Allescheria  Gayoni  (Eurotiopsis  Gayoni)j  which  produces 
zymase  during  a  life  very  much  more  aerobic  than  that  of  the  yeast 
and  which  always  gives  a  little  alcohol  in  the  presence  of  air  which, 
moreover,  it  uses  for  food.  In  Raulin's  medium,  in  which  alcohol  is 
substituted  for  sugar,  the  fungus  vegetates  very  easily.  Alcohol  is, 
then,  a  useful  substance  for  it.  It  disappears  in  the  form  of  water  and 
carbon  dioxide  with  a  rapidity  comparable  to  that  of  carbohydrates. 
The  alcohol  may  be  regarded  as  an  intermediary  product  in  the  metab- 
olism of  the  sugars.  It  is  not  apparent  because  it  is  used  up  as  rap- 
idly as  it  is  formed.  Quite  recent  experiments  of  Trillat  and  Sauton, 
as  well  as  those  of  Kayser  and  Demlon,  have  shown  that  after  the 
complete  disappearance  of  sugar  in  wine,  the  yeast  acts  like  any  ordi- 
nary cell.  In  presence  of  air  it  respires  like  ordinary  plants  by  oxidizing 
organic  acids.  It  may  oxidize  the  alcohol,  and  when  agitated  in  the 
presence  of  air,  ethyl  or  acetic  aldehydes  may  be  formed  by  this  oxi- 
dation. It  acts,  then,  like  Allescheria  Gayoni  but  in  a  more  active 
manner.  On  the  other  hand  it  has  been  known  for  a  long  time  that 
many  of  the  Mycoderma,  especially  Mycoderma  vini,  and  the  myco- 
yeast  of  Duclaux,  are  capable  of  maintaining  themselves  at  the  ex- 
pense of  alcohol.  A.  Perrier  l  has  encountered  a  certain  number  of 
microorganisms  endowed  with  a  considerable  oxidizing  power  and  in 
particular  capable  of  developing  in  a  mineral  medium  containing  ethyl 
aldehyde  as  the  source  of  carbon.  This  assembly  of  facts  seems  then 
to  prove  that  alcohol  and  aldehyde  are  able  to  represent  two  stages 
in  the  assimilation  of  carbohydrates  by  plants. 

1  Perrier,  A.  Sur  la  combustion  de  Taldehyde  ethylique  par  les  vegetaux 
inferieurs.  Comp.  Rend.  Acad.  Sciences,  151,  1910. 


104  PHYSIOLOGY  OF  YEASTS 

This  theory  has  the  advantage  of  explaining  the  liberation  of  car- 
bon dioxide  and  the  accumulation  of  alcohol  during  asphyxia  of  a 
plant  deprived  of  oxygen. 

However,  numerous  objections,  in  spite  of  the  illustrations  which 
have  been  presented,  have  been  raised  that  alcohol  is  rarely  a  food  for 
the  yeasts.  The  oxidation  of  alcohol  by  fungi  is  not  able  to  be  re- 
garded as  a  rare  occurrence.  According  to  certain  authors,  alcohol 
seems  to  be  a  waste  product.1 

Kostytschew  2  has  noticed  that  when  wheat  grains  are  kept  away 
from  air  and  finally  placed  in  air,  alcohol  is  produced  during  the  fer- 
mentation and  not  the  oxide.  Certain  investigators  have  added  to  this 
theory  a  modification  which  resolves  some  of  the  difficulties.  Thus 
Kostytschew,  Boy  en-Jensen,3  and  Blackmann 4  have  admitted  that 
alcohol  produced  by  fermentation  is  not  oxidizable.  Alcohol  will 
not  be  a  normal  product  of  respiration;  it  will  form  only  when  the 
intermediary  products  of  respiration  escape  the  action  of  oxydases. 
According  to  Boyen-Jensen  and  Blackmann  the  true  intermediary 
product  will  be  dioxyacetone.  Kusserow  5  considers  alcoholic  fermen- 
tation in  the  light  of  incomplete  respiration.  With  the  exclusion  of 
air  the  yeast  reduces  the  sugar  to  a  diatomic  alcohol  which  further 
reduces  to  ethyl  alcohol,  carbon  dioxide  and  hydrogen. 


Autophagy  or  Autolysis  of  Yeasts 

We  should  now  investigate  the  curious  phenomenon  known  as 
autophagy  or  autolysis.  When,  in  a  fermentation,  the  quantity  of 
yeast  is  lower  than  40  per  cent  by  weight  of  the  sugar,  the  fermenta- 
tion stops  immediately  at  the  exhaustion  of  the  sugar.  But  if,  on  the 
contrary,  the  quantity  of  yeast  is  greater  than  40  per  cent  of  the  sugar 
by  weight,  the  fermentation  continues  after  the  exhaustion  of  the  sugar. 
The  yeast  lives,  then,  on  its  own  substances.  It  ferments  the  glyco- 
gen  which  it  has  accumulated  and  accomplishes  a  sort  of  autodigestion. 

This  phenomenon  may  be  observed  in  a  yeast  undergoing  inani- 

1  Maquenne,    L.     La  respiration  des  plantes  vertes.     Rev.  g.   des   Sciences, 
19Q5.     16th  year.    No.  13. 

2  Kostytschew,    S.      Ueber   ein   Einfluss   ergorgener   Zuckerlosungen   auf   die 
Atmung  der  Weizenkeime.    Bioch.  Zeitschr.  23,  1910. 

3  Boyen-Jensen,  P.     Die  Zersetzung  des  Zuckers  wahrend  des  Respiration  Pro- 
zesses.    Ber.  der  deutschen  Bot.  Ges.  26,  1909. 

4  Blackmann,  F.     Assoc.  Brit,  pour  1'Av.  des  Sciences.     Congres  de  Scheefield, 
1910. 

6  Kusserow,  R.  Respiration  and  fermentation,  two  allied  physiological  proc- 
esses. Brennerci  und  Pressehefefabrik,  44  (1912),  1-3;  Chemical  Abstracts,  7 
(1913),  2237. 


AUTOPHAGY  OR  AUTOLYSIS  OF  YEASTS  105 

tion  in  water  to  which  a  little  antiseptic  has  been  added  (creosote, 
toulol,  phenol,  etc.).  The  yeast  is  reduced  to  live  at  the  expense  of 
its  glycogen  which  it  has  laid  up  in  reserve  and  to  attack  its  protein 
substances.  Then,  two  phases  may  be  distinguished  in  autophagy, 
first,  the  fermentation  of  glycogen  and,  secondly,  the  proteolysis  of 
its  own  protein  substances. 

Glycogenase  which  is  contained  in  the  cells  acts  on  the  glycogen 
and  causes  a  fermentation  (autof ermentation) .  The  principal  prod- 
ucts formed  under  these  conditions  are  alcohol,  carbon  dioxide, 
glycerol,  and  according  to  Salkowski,  succinic  acid.  The  fermenta- 
tion of  glycogen  is  then  quite  analogous  to  intramolecular  respira- 
tion which  is  noticed  in  fruits  containing  sugar  when  placed  away 
from  air. 

The  yeast,  at  the  same  time,  attacks  albuminoid  materials  by 
means  of  its  endotryptase  and  other  proteases  (guanase  and  argin- 
ase)  which  seem  to  play  a  very  important  role.  The  products  of 
this  digestion  are  nucleic  bases,  tyrosine,  leucine,  guanine,  lysine, 
arginine,  aspartic  acid  (Kutscher)  and  choline.  Autophagy  depends, 
according  to  Effront,  not  on  the  cells  but  on  the  enzymes  which  are 
formed  in  the  cells  when  placed  in  inanition.  In  the  presence  of  water 
the  hydroxy  compounds  of  carbon  disappear  with  the  liberation  of 
carbon  dioxide.  The  cells  die  rapidly  at  the  end  of  about  6  days. 
In  the  presence  of  alcohol  (7  per  cent)  and  a  little  hydrofluoric  acid, 
Effront l  has  been  able  to  obtain  a  proteolysis  of  albuminoid  sub- 
stances; under  these  conditions  the  yeasts  are  easily  able  to  support 
a  denutrition  without  losing  their  fermenting  power. 

1  Effront,  J.    Sur  F  autophagy  de  la  levure.    Moniteur  scientific,  1905,  16. 


CHAPTER  IV 
PHYSIOLOGY   OF    YEASTS    (CONTINUED) 

Living  Conditions  of  Yeasts.     Their  Relations  to  Their  Environ- 
ment.   Parasitism  and  Symbiosis 

IN  this  chapter  we  shall  consider  the  living  conditions  of  yeast, 
i.e.,  their  habitat,   their  duration   of   life,   the   physico-chemical 
conditions  which  are  necessary  for  their  development,   the  in- 
fluences  which    determine    budding,    the    production    of   spores,   and 
finally,  the  pathogenic  yeasts  and  the  question  of  symbiosis. 

Habitat  of  Yeasts 

Devoid  of  chlorophyll,  the  yeasts,  like  all  other  fungi,  are  unable 
to  assimilate  atmospheric  carbon.  They  are,  then,  necessarily  para- 
sites or  saprophytes.  A  certain  number  among  them,  such  as  the  beer- 
yeasts  and  industrial  yeasts,  the  cultivated  yeasts,  have  been  prop- 
agated from  time  immemorial  by  man.  The  greater  part  of  them  live 
saprophytically,  especially  when  sugar  is  available.  These  are  able  to 
be  regarded  to  a  certain  extent  as  domestic  yeasts.  In  certain  regions 
fruits  make  a  good  environment  on  account  of  their  sugar  content. 
However,  a  nectar  may  also  be  found  in  certain  flowers  (Berlese),1  in 
the  mucous  secretions  of  trees  (Ludwig,  Hansen,2  Lindner,  Rose 3) 
and  rarely  in  the  detritus  from  vegetable  decomposition.  It  will  be 
pointed  out  later  on,  that  at  the  end  of  autumn,  the  yeasts  are  intro- 
duced into  the  soil  by  the  fall  of  the  fruits  and  the  rains  and  there 
pass  the  winter. 

The  investigations  of  De  Kruyff  have  shown  that,  contrary  to  the 
conditions  in  Europe,  the  yeasts  in  Java  are  very  much  more  distrib- 
uted on  the  leaves,  both  living  and  dead.  The  exceptional  climato- 
logical  conditions  of  this  country,  humidity  and  heat,  favor  this  mode 
of  life.  Many  Torula,  Mycoderma  and  a  few  true  yeasts  have  been 

1  Berlese,  A.     Verhalten  der  Sacch.  an  den  Weinstocken.       Rivista  di  pat. 
vegetal!,  5,  1897. 

2  Hansen,   E.   C.     Ueber  die  im  Schleimflusse  lebenden  Baume  beobachten 
Mikroorganismen.    Cent.  Bakt.  5,  1889. 

3  Rose,   L.     Beitrage  zur  Kenntniss  der  Organismen  im  Eichenschleimfluss. 
Inaugural  Dissert.  Universitat  Berlin.    25  June,  1910. 

106 


HABITAT  OF   YEASTS  107 

found  in  the  salt  waters  of  the  sea  (Fischer  and  Brebeck  ).1  They 
are  frequently  encountered  in  milk.  (Maze,  Dombrowski.)  Finally 
many  of  the  Mycoderma  live  in  alcoholic  beverages. 

Many  of  the  yeasts  live  as  parasites  in  man  and  animals  and  cause 
quite  varied  lesions. 

Duration  of  Life  of  Yeasts 

The  yeasts  are  capable  of  conserving  themselves  for  a  long  time  in 
the  same  media  without  perishing.  Duclaux,2  who  has  given  this 
subject  some  attention,  had  the  privilege  of  studying  some  of  the  cul- 
tures which  Pasteur  had  used  in  his  investigations  on  alcoholic  fer- 
mentation. In  1885,  after  5  or  6. years,  in  15  attempts  on  old  yeasts, 
he  found  only  three  which  had  died  out.  In  1889,  after  11  to  17 
years,  out  of  26  yeasts  only  6  could  not  be  revived.  He  has  been  able 
to  observe  living  yeasts  after  25  years.  From  this  it  will  be  seen 
that  the  yeasts  are  able  to  live  for  a  long  time  in  media  in  which  the 
ordinary  foods  have  been  exhausted  and  to  use  materials  which  or- 
dinarily would  not  be  taken.  Hansen  found  that  yeasts  could  be  kept 
for  periods  of  from  13  to  17  years  in  a  liquid  containing  10  per  cent 
of  sucrose  without  acid. 

Recent  observations  by  Klocker3  at  the  laboratory  in  Carlsberg 
have  shown  that  living  cells  of  yeast  were  present  in  sucrose  and  beer 
wort  solutions  after  20  and  30  years.  Will 4  has  also  found  that  yeasts 
would  live  for  a  long  time  in  media  such  as  beer  wort.  Among  the 
cultures  which  were  examined,  the  oldest  was  18  years  and  2  months. 

Will 5  has  published  data  on  the  longevity  of  yeasts  under  different 
conditions.  He  found  that  the  yeasts  would  live  longer  in  liquid  gela- 
tin because  these  remained  moist  longer.  Even  in  dry  cultures  there 
would  be  living  cells  after  a  long  time.  Meissner 6  secured  some 

1  Brebeck,  B.   and  Fischer,   B.     Zur  morph.   Biol.   und  Syst.  d.  Kahmpilze, 
Jena,  1891. 

2  Duclaux,  E.      Traite    de    Microbiologie,   Fermentation   alcoolique   3,  Paris, 
1900. 

3  Klocker,   A.     Fermentation  organisms.     III.   Conservation  of  fermentation 
organisms  in  different  nutritive  media.      Comp.  Rend.  trav.  lab.  Carlsberg,    11 
(1917),  297-311. 

4  Will,  H.    Noch  einige  Mitteilungen  iiber  das  Vorkommen  von  lebens-  und 
vermehrungsfahigen  Zellen  in  alten  Kulturen  von  Sprosspilzen.     Cent.  Bakt.  48 
(1917),  35-41. 

6  Will,  H.  The  persistence  of  living  yeast  on  gelatin  cultures.  Cent.  Bakt. 
Abt.  II,  31,  436-453;  Beobachtungen  liber  das  Vorkommen  lebens-  und  vermeh- 
rungsfahigen Zellen  in  sehr  alten  Wiirzkulturen.  Cent.  Bakt.  Abt.  II,  44,  1916, 
58-75. 

6  Meissner,  R.  Ten-year  experiment  on  the  longevity  of  wine  yeasts  in  pure 
cultures  on  10  per  cent  cane  sugar  solutions.  Zeit.  Garungsphysiologie,  1,  106-113. 


108  PHYSIOLOGY  OF  YEASTS   (Continued) 

interesting  data  on  the  longevity  of  yeasts.  He  used  25  pure  cul- 
tures grown  in  10  per  cent  cane  sugar  solutions  without  renewal. 
Fifteen  of  these  yeasts  retained  their  vitality  for  10J  years,  while 
nine  of  them  died  after  eight  and  one-half  years.  One  remained  alive. 
Gayon  and  Dubourg  1  made  an  investigation  which  bears  indirectly 
on  this  subject.  They  examined  wines  made  in  1810,  1818,  1819,  1832, 
1836  and  1846  and  found  living  yeasts  capable  of  causing  alcoholic 
fermentation. 


ACTION  OF  PHYSICAL  AGENTS  ON  THE  YEASTS 

Temperature 

Moist  yeasts  die  generally  between  50°  and  55°  C.  some  being  able 
to  withstand  60°  (Hansen  and  Kayser2).  In  the  dry  state,  they  re- 
sist more  elevated  temperatures.  Certain  are  able  to  withstand,  with- 
out perishing,  a  temperature  of  from  100  to  110°,  others  from  115  to 
120°.  The  ascospores  are  much  more  resistant  to  heat  and  generally 
withstand  a  temperature  which  is  about  5°  higher  than  the  vegetative 
cell.  (Kayser.)  On  the  other  hand,  the  investigations  of  Pictet  and 
Young  indicate  that  the  yeasts  are  capable  of  resisting  very  intense 
cold.  These  investigators  have  submitted  yeasts  to  temperatures  of 
130°  below  zero  for  24  hours  without  killing  them.  Doemus  has  stated 
that  the  yeast  of  Frohberg  could  resist  temperatures  of  —  150°  for 
from  5  to  20  minutes.  Cochran  and  Perkins  3  investigated  the  effect 
of  high  temperatures  on  yeast.  They  heated  their  yeasts  in  a  syrup 
and  found  that  58°  C.  for  30  minutes  did  not  stop  fermentation;  65°  C. 
for  the  same  length  'of  time  caused  a  devitalization  of  the  yeast  so 
that  fermentation  was  reduced.  The  yeasts  were  killed  at  70°  C. 
Wells  4  found  that  the  thermal  death  point  in  yeasts  is  considerably 
effected  by  the  presence  of  certain  substances.  Starch  and  sugars 
were  found  to  raise  it.  He  states  that  the  approximate  thermal  death 
point  in  bread  is  about  68°  C.  It  is  quite  well  known,  however,  that 
bread  may  contain  living  cells  since,  during  the  baking  process,  the 
temperature  is  not  high  enough  to  kill  all  of  the  yeasts. 

1  Gayon  and  Dubourg.     Experiments  on  the  vitality  of  yeasts.     Rev.  vit. 
38,  5-8. 

2  Kayser,  E.    Action  de  la  chaleur  sur  les  levures.     Ann.  Inst.  Past.  3,  1889. 
s  Cochran,  C.  B.  and  Perkins,  J.  H.     The  effect  of  high  temperatures  on 

yeast.    Jour.  Ind.  Eng.  Chem.  6  (1914)  480. 

4  Wells,  E.  P.  The  thermal  death  point  in  yeast.  Vermont  Agricultural  Ex- 
periment Station,  Bull.  203,  1917. 


ACTION   OF  PHYSICAL  AGENTS   ON   THE  YEASTS     109 

Light 

Light  does  not  seem  to  possess  any  marked  action  on  yeasts.  How- 
ever, Marshall  Ward  1  noticed  a  destructive  action  of  light  on  the 
ascospores  of  S.  Pyriformis.  It  will  be  pointed  out  that  the  different 
rays  of  the  spectrum  have  an  accelerating  or  retarding  influence  on 
the  sporulation  of  yeasts.  According  to  Martinand  yeasts  are  de- 
stroyed by  an  exposure  of  4  hours  to  the  sun's  rays  at  40  to  45°. 
An  exposure  of  3  days  at  36°  produces  the  same  effect.  The  absence 
of  light,  on  the  contrary,  over  a  long  time  does  not  effect  the  yeasts. 
Although  light  acts  on  the  vitality  of  the  yeasts,  it  does  not  seem  that 
its  effect  is  very  great,  and  it  may  be  generally  said  that  yeasts  are 
resistant  to  the  action  of  light. 

The  recent  investigations  of  Buchta 2  made  with  Saccharomyces 
cerevisiae  and  Ludwigii  have  shown  that  diffuse  daylight  stopped  the 
budding  of  yeasts.  The  cells  not  exposed  to  it  multiplied  almost  twice 
as  fast  as  those  which  were  exposed.  Electric  light  had  the  same  in- 
fluence. When  cultures  of  yeasts  were  placed  at  different  distances 
from  the  source  of  light,  those  which  were  farthest  away  multiplied 
most  quickly.  The  blue  light  had  a  more  marked  action  than  the 
red  which  did  not  seem  to  effect  the  budding.  The  infra-red  rays  did 
not  seem  to  impede  budding.  The  ultra-violet  rays,  however,  ex- 
hibited a  marked  action,  and  an  exposure  of  10  seconds  sufficed  to 
stop  budding.  A  longer  time  resulted  in  the  death  of  the  cells.  Von 
Recklinghausen  3  found  that  it  took  300  seconds'  exposure  at  a  dis- 
tance of  200  mm.  from  a  quartz  lamp  burning  66  volts  and  3.5  am- 
peres to  kill  yeast  cell. 

Jacquemin  and  Giurel 4  found  that  radioactive  emanations  exerted 
a  favorable  action  on  fermentation.  A  radioactivity  of  \  to  1  unit 
per  liter  exerted  a  favorable  action  on  the  splitting  of  sugars. 

Moisture 

Yeasts  need  moisture;  they  resist  drying  easily  as  the  experiments 
of  Hansen 5  have  shown. 

1  Ward,   H.   M.     The  ginger  beer  plant  and  the  organisms  composing  it. 
Philos.  Trans.  Royal  Soc.,  1898,  183. 

2  Buchta,  L.     Ueber  den  Einfluss  des  Lichtes  auf  die  Sprossung  der  Ilefe. 
Cent.  Bakt.  Abt.  II.  41  (1914),  340. 

3  Von  Recklinghausen,    M.     Purification  of  water  by  the  ultraviolet  rays. 
Jour.  Amer.  Water  Works  Assn.  1  (1914),  565-588. 

4  Jacquemin,  G.  and  Giurel,  G.     The  influence  of  radioactive  emanations  on 
yeasts  and  alcoholic  fermentations.    Bull.  Agr.  Intelligence,  5  (1914),  1505. 

5  Hansen,  E.  C.     Recherche  sur  la  morph.  et  la  physiol.  des  ferments  al- 
cooliques.    Comp.  Rend,  des  trav.  de  Carlsberg,  4,  1898. 


110  PHYSIOLOGY  OF   YEASTS   (Continued) 


Metals  and  Salts 

Zikes  1  found  that  aluminum  had  a  slightly  stimulating  effect  on 
the  fermenting  and  regenerative  function.  Bokorny 2  studied  the 
effect  of  certain  uncommon  salts.  RB2SO4  and  Cs2SO4  in  the  presence 
of  potassium  salts  favored  the  development  of  yeast.  Lithium  salts 
proved  injurious  to  yeast  propagation  An  increase  of  over  0.1  per 
cent  of  the  potassium  phosphates  in  a  medium  is  not  advantageous. 
Two  per  cent  (NH4)2S04  does  not  seem  to  hinder  the  development 
of  yeast.  Bokorny,3  in  another  paper,  has  reported  a  very  complete 
study  of  the  action  of  metallic  salts.  Practically  all  of  the  metallic 
salts  were  studied.  Kossowicz  4  has  shown  that  yeasts  liberate  iodin 
from  Kl-mineral-sugar  solutions. 

Bokorny  5  has  stated  that  manganese  is  not  poisonous  to  yeasts 
if  it  is  in  the  form  of  its  salts.  Budding  took  place  when  yeast  was 
put  into  1  per  cent  solution  of  MnSO4,  while  in  a  3  or  5  per  cent  solu- 
tion budding  was  stopped.  This  is  attributed  to  the  fact  that,  unlike 
the  other  metals,  manganese  does  not  unite  with  the  protoplasm. 
Boas  6  studied  the  effect  of  arsenic  compounds  on  yeast.  He  found 
that,  at  first,  sodium  metaarsenite  and  potassium  and  sodium  arsenate 
had  a  repressing  action  which  was  eventually  overcome  if  the  yeast 
was  kept  in  contact  with  these  solutions  for  a  period  of  time.  Low 
temperatures  were  said  to  increase  the  intensity  of  the  poisoning  action 
but  without  killing  the  yeast.  Mitra7  found  that  the  chloride  of 
sodium,  potassium J  calcium  and  magnesium  are  more  or  less  toxic 
to  yeasts  in  concentrations.  KC1  was  the  least  ?nd  NaCl  the  most 
harmful. 

1  Zikes,  H.     Influence  of  aluminum  on  yeast  and  beer.    Allgem.   Zeit.    Bier- 
brau.  Malzfabr.  41,  71-4,  83-7. 

2  Bokorny,   Th.     The  influence  of   cesium,   rubidium,   and  lithium  salts  on 
yeast  in  comparison  with  the  action  of  potassium  and  ammonium.     Allg.  Braw. 
Hopfen-Ztg.  52,  1469-70. 

3  Bokorny,  Th.     Action  of  salts  of  metals  upon  yeast  and  other  fungi.     Cent. 
Bakt.  Abt.  II.,  35,  118-197. 

4  Kossowicz,  A.  and  Loew,  W.     The  behavior  of  bacteria,  yeasts  and  molds 
towards  iodin  compounds.    Z.  Garungsphysiologie,  2,  1913. 

5  Bokorny,   Th.     The  non-poisonous  properties  of   manganese.     Chem.  Ztg. 
38,  1290. 

6  Boas,  F.     Action  of  arsenic  compounds  on  yeast.     Chemical  Abstracts,  12 
(1918),  1101. 

7  Mitra,  S.  K.    Toxic  and  antagonistic  effects  of  salts  on  wine  yeasts.     Calif. 
Pub.  Agr.  Sci.  (15)  3,  63-102,  1917. 


PRESSURE  111 

Pressure 

The  investigations  of  Regnard l  and  Melsens 2  have  shown  that 
the  yeasts  are  able  to  resist  a  very  strong  pressure.  Regnard  submitted 
a  yeast  to  a  pressure  of  1000  atmospheres  for  1  hour  without  killing 
it.  '  Melsens  has  used  pressures  of  8000  atmospheres  and  noticed  no 
diminution  in  the  vitality  of  the  yeasts  so  treated.  Hite,  Giddings 
and  Weakley  3  in  studying  the  effects  of  high  pressures  on  micro- 
organisms used  a  number  of  common  yeasts.  Samples  of  grape 
sugar  in  water  which  were  inoculated  with  baker's  (Fleischman's) 
yeast  did  not  ferment  after  being  subjected  to  60,000  pounds  pres- 
sure per  square  inch  for  a  half  hour  at  room  temperature.  With 
Saccharomyces  cerevisiae,  Meyer,  there  was  no  growth  after  80,000 
pounds  pressure.  While  the  results  are  not  concordant,  it  may  be 
seen  that  this  yeast  could  stand  pressures  of  between  50,000  and 
55,000  Ibs.  pressure  for  10  or  20  minutes,  in  distilled  water.  In  3  per 
cent  cane  sugar  solution,  there  was  one  instance  where  this  yeast  re- 
sisted 60,000  pounds  for  10  minutes.  Saccharomyces  albicans,  Reess, 
in  one  instance,  resisted  60,000  pounds  pressure  for  10  minutes.  Chop- 
lin  and  Tammann,4  as  quoted  by  Hite  and  his  colleagues,  have  stated 
that  yeasts  could  resist  a  pressure  of  3000  kilograms  per  square  centi- 
meter (43,000  pounds  per  square  inch). 

Antiseptics 

Nowak  5  found  that  ozonization  had  a  detrimental  effect  on  the 
multiplication  of  yeast.  It  was  brought  out  that  this  method  may 
be  used  to  remove  undesirable  bacteria  from  yeast  cultures.  Lind- 
ner and  Grouven  6  employed  four  disinfectants  towards  yeast.  These 
were  corrosive  sublimate  (ammonium),  fluoride,  formalin,  and  anti- 
formin.  Will  and  Wieninger 7  experimented  with  ozone  on  yeast. 

1  Regnard,  P.    Influence  de  la  pression  sur  la  levure.    C.  R.  Ac.  des  Sciences, 
98,  1884. 

2  Melsens.    Comp.  Rend.  Ac.  des  Sci.  1870. 

3  Hite,  B.  H.,  Giddings,  N.  J.,  and  Weakley,  C.  E.     The  effect  of  pressure 
on  certain  microorganisms  encountered   in   the   preservation  of    fruits   and  vege- 
tables.    Bulletin  146,  West  Virginia  University  Agricultural  Experiment  Station, 
1914. 

4  Choplin,  G.  W.  and  Tammann,  G.     Ueber  den  Einfluss  hoher  Eindruck  an 
Mikroorganismen.    Zeit.  Hygiene,  45  (1903),  171. 

6  Nowak,  C.  A.  Influence  of  ozone  on  yeast  and  bacteria.  Jour.  Ind.  Eng. 
Chem.  5,  668,  1913. 

6  Lindner,  P.  and  Grouven,  O.    What  influence  has  an  increase  in  the  quantity 
of  yeast  on  the  disinfecting  power  of  various  antiseptics.     Wochschr.  Brau.  30, 
133-135. 

7  Will,  H.  and  Wieninger.     Zeit.  f.  d.  ges.  Brau..     1910. 


112  PHYSIOLOGY  OF  YEASTS   (Continued] 

They  found  that  0.56  gram  in  a  cubic  centimeter  of  air  was  toxic 
for  30,000,000  cells. 

Euler  and  Emberg  l  have  stated  that  the  hydrogen  centration  in- 
fluences the  development  of  bottom  yeast.  This  is  to  be  expected 
since  other  forms  of  microorganisms  act  in  the  same  way. 


Physiological  Conditions  of  Budding 

Budding  is  accomplished  each  time  the  cell  finds  itself  in  a  suit- 
able environment  with  no  deterring  factors,  such  as  the  accumulation 
of  products  of  metabolism.  Aeration  plays  an  important  role;  it 
seems  to  accelerate  budding.  However,  it  is  not  indispensable.  Han- 
sen  2  has  noticed  that  budding  took  place  in  the  presence  of  nitrogen 
with  no  oxygen  present.  It  is  also  known  that  budding  continues 
during  alcoholic  fermentation.  It  is  known,  to  the  contrary,  that 
sporulation  does  demand  the  presence  of  oxygen.  Sporulation  and 
budding  then  differ  on  this  point  which  closely  separates  them. 

Temperature  exerts  a  preponderant  influence  on  budding  which 
is  a  function  of  temperature.  Hoyer  has  calculated,  for  example,  that 
in  a  medium  of  gelatin,  S.  Pastorianus  formed  a  new  generation  at 
13°  every  (5  hours  while  at  35°  the  budding  was  accomplished  in  about 
3  hours.  The  experiments  of  Hansen  have  shown  that  there  exist 
minimum,  optimum  and  maximum  temperatures  for  every  species  of 
yeasts.  Hansen  has  determined  the  limiting  temperatures  for  11 
species  of  yeasts  (S.  cerevisiae,  Pastorianus,  intermedius,  validus, 
ellipsoideus,  turbidans,  Marxianus,  Willia  anomala,  Pichia,  membranae- 
faciens,  Saccharomy  codes,  Ludwigii).  He  found  that  the  maximum 
temperatures  of  these  species  varied  from  47°  to  34°  C.,  the  minimum 
temperature  from  0.5°  to  0.3°  C. 

Particular  Types  of  Budding 

It  has  been  stated  above  that  yeasts  may  grow  as  a  sediment  at 
the  bottom  of  the  culture  flasks  (anaerobic  life),  or  as  a  scum  or  veil 
(aerobic  life).  The  formation  of  the  scum  generally  represents  a  par- 
ticular type  of  budding. 

1  Euler,  H.  and  Emberg,  F.     The  sensitiveness  of  living  yeast  to  hydrogen  and 
hydroxyl-ion    concentration.      Zeit.    Biol    69,   349-64   1919.     Chem.    Absts.     13 
(1919)  2691. 

2  Hansen,  E.  C.    Recherches  sur  la  physiol.  et  la  morphologic  des  alcooliques 
ferments  XI.     La  spore  de  Saccharomy ces  de venue  sporange.     Recherches  com- 
paratives  sur   les   conditions   de   croissance   vegetative    et    le    development    des 
organes  de  reproduction  des  levures  et  des  moisissures.     C.  R.  du  lab.  de  Carlsb. 
5,  Book  2,  1902. 


PARTICULAR  TYPES  OF  BUDDING  113 

Certain  yeasts,  such  as  the  Mycoderma  (Mycoderma  vini  and  cere- 
visiae),  are  essentially  aerobes,  never  producing  fermentations  and 
forming  on  the  surface  of  the  media  a  scum  which  is  very  character- 
istic, reminding  one  of  fungi.  This  scum  is  gray  and  dry.  Later,  it 
develops  and  becomes  wrinkled.  Many  bubbles  of  air  are  found  re- 
tained between  the  cells.  But  the  Mycoderma  are  not  characteristic 
yeasts;  they  do  not  form  spores  and  their  place  in  a  classification  is 
quite  uncertain. 

Hansen  has  distinguished  two  groups  among  the  Saccharomycetes 
or  true  yeasts.  In  one,  which  includes  Willia  and  Pichia,  the  scum 
appears  very  rapidly  at  the  beginning  of  the  culture.  It  is  well  devel- 
oped, dry,  and  filled  with  globules  of  air  which  are  retained  between 
the  cells.  This  is  the  characteristic  scum  for  the  Mycoderma.  For 
this  group,  the  scum  is  a  normal  method  of  vegetation.  It  is  under- 
stood, then,  that  budding  is  confused  with  the  formation  of  the  scum. 

In  the  other  group,  to  which  belong  the  majority  of  known  species, 
a  scum  may  or  may  not  be  formed.  When  one  is  formed  it  appears 
at  the  end  of  fermentation  and  under  conditions  which  Hansen  has 
well  established.  On  the  other  hand,  this  scum  differs  in  the  group. 

It  is  necessary,  in  order  for  the  scum  to  form,  that  the  surface 
of  the  medium  be  quiet  and  in  direct  contact  with  air.  Hansen  has 
recommended  that  a  24-hour  culture  be  used  to  which-  there  is  added 
new  wine.  The  culture  should  be  shaken,  after  which  a  drop  is  carried 
over  to  a  flask  half  full  of  new  wine  and  closed  with  a  cap  of  paper. 
At  the  end  of  a  shorter  or  longer  time,  the  principal  fermentation  is 
finished  and  on  the  surface  of  the  medium  are  seen  little  spots  of 
yeast.  These  remain  isolated  as  little  islands,  until  they  join  to  form 
a  thin  scum  which  is  gray  and  mucous.  If  the  flask  is  shaken,  parts 
of  the  scum  break  off  and  fall  to  the  bottom;  eventually  a  new  scum 
will  form  over  the  surface.  During  the  formation  of  this  scum,  the 
medium  becomes  a  clear  yellow.  The  scum,  thus  formed,  differs 
markedly  from  that  formed  by  Mycoderma;  it  is  less  tenacious  and 
has  a  more  viscous  appearance. 

Other  yeasts  do  not  form  scums  but  simply  rings  about  the  side 
of  the  tube.  With  some  both  are  formed. 

The  formation  of  the  scum  is  influenced  by  the  temperature,  as 
the  investigations  of  Hansen  have  determined.  Hansen  has  shown  that 
certain  limits  of  temperature  exist  outside  of  which  the  scum  is  not 
able  to  form.  Between  these  limits  the  time  of  formation  is  determined 
by  the  temperature.  The  time  is  constant  for  a  given  temperature. 
A  certain  optimum  temperature,  variable  with  each  species,  allows  the 
most  rapid  formation  of  the  scum.  It  is  an  interesting  fact  to  note 
that  there  is  no  relation  between  the  temperature  at  which  scum  for- 


114  PHYSIOLOGY  OF   YEASTS   (Continued) 

mation  goes  on  most  rapidly  and  budding.    Budding  is  less  dependent 
on  the  temperature. 

In  determining  these  various  temperatures  and  the  time  necessary 
for  the  scum  to  form  in  six  species  (S.  cerevisiae,  Pastorianus,  inter- 
medius,  validus,  ellipsoideus,  and  turbidans),  Hansen  noticed  that  scum 
formation  was  slower  at  low  temperatures  than  at  high  temperatures. 
On  the  other  hand,  it  may  be  stated  that  the  maximum  temperature 
for  scum  formation  is  lower  than  the  maximum  for  budding.  The 
temperature  limits  of  scum  formation  vary  with  the  species  and  fur- 
nish important  characteristics  for  the  differentiation  of  these  species. 

Physiological  Conditions  of  Sporulation 

We  shall  see,  in  a  later  chapter,  that  sporulation  is  generally  a 
function  of  inanition  of  the  yeast.  It  is  often  necessary  that  the 
yeasts  have  accumulated  from  former  culture  media  the  reserve  prod- 
ucts necessary  for  the  formation  of  ascospores.  Under  these  condi- 
tions, the  cells  begin  to  bud  as  soon  as  these  reserve  products  are 
exhausted.  It  seems  from  all  this  that  the  formation  of  ascospores 
is  determined  exclusively  by  the  lack  of  food.  This  is  the  conclusion 
to  which  the  investigations  of  Klebs  l  leads  us.  However,  it  has 
been  known  for  a  long  time  that  the  yeasts  are  able  to  sporulate  very 
rapidly  on  certain  solid  media  (gelatin  added  to  wine,  slices  of 
carrot  or  potato)  and  sometimes  in  liquid  media  during  fermentation. 
Sporulation  seems,  then,  to  have  other  causes.  Klebs  admits  that  in 
the  case  of  solid  media,  such  as  nutrient  gelatin  or  slices  of  carrot,  if  the 
yeasts  are  able  to  sporulate,  it  is  only  those  cells  which  are  in  the 
innermost  parts  of  the  colonies  where  they  are  prevented  from  using 
the*  medium.  These  find  themselves  in  bad  conditions  of  food  supply 
which  explains  their  sporulation.  In  these  colonies,  the  cells  occupy- 
ing the  marginal  portion  of  the  colony  will  continue  to  bud  and 
multiply  while  the  cells  which  are  on  the  inside  of  the  colony  will  be 
reduced  to  conditions  which  favor  the  formation  of  spores. 

The  investigations  of  Hansen,2  to  which  we  owe  much  of  our  in- 
formation with  regard  to  sporulation,  have  demonstrated,  on  the  con- 
trary, that  this  matter  is  much  more  complicated.  If  the  lack  of 
food  is  one  of  the  most  important  factors,  it  is  by  no  means  indis- 
pensible.  Indeed,  Hansen  has  stated  that  contrary  to  the  ideas  of 
Klebs,  in  cultures  on  gelatin  or  beer  wort,  the  ascospores  form  in  the 

1  Klebs,  C.    Allgemeine  Betrachtungen.     Jahrb.  wissenschaft.  Bot.  35,  1900. 

2  Hansen,  E.  C.     Recherches  sur  la  physiologic  et    la  morphologic    des  fer- 
ments alcooliques.     II.  Les  ascospores  chez  le  genre  Saccharomyces.    III.  Sur  les 
Torula  de  M.  Pasteur.    IV.  Maladies  provoquees  dans  la  biere  par  les  ferments 
alcooliques.    C.  R.  du  lab.  de  Carlsberg,  1,  1883,  5,  1902. 


PHYSIOLOGICAL  CONDITIONS  OF  SPORULATION     115 

cells  on  the  margin  as  well  as  in  the  cells  inside  of  the  colonies.  The 
lack  of  food,  then,  has  little  bearing  in  this  case  because  the  well-nour- 
ished cells  also  form  ascospores. 

According  to  Hansen,  two  factors  seem  to  determine  sporulation: 
the  lack  of  food  and  the  accumulation  in  the  medium  of  toxic  excre- 
tion of  the  yeast  cell.  With  the  yeasts  which  are  placed  on  gypsum 
blocks  or  in  distilled  water,  it  is  the  lack  of  food  which  is  probably 
the  reason  for  sporulation.  With  yeasts  cultivated  on  solid  media 
(slices  of  carrots,  or  nutrient  gelatin)  it  is  the  action  of  toxic  excre- 
tions which  arrests  budding  and  causes  sporulation.  It  is  the  same 
reason  which  causes  some  yeasts  to  form  ascospores  in  a  fermenting 
solution.  The  alcohol  may  hinder  budding  and  provoke  sporulation. 
Hansen  has  shown,  however,  that  certain  chemical  substances,  such  as 
saturated  calcium  sulfate,  are  capable  of  stopping  budding  and  pro- 
ducing sporulation. 

In  recent  investigations  by  Saito,  the  role  of  toxic  substances  of 
the  yeast  in  relation  to  sporulation  has  been  studied.  According  to 
this  author,  only  the  cells  on  the  periphery  of  a  colony  sporulate. 
It  seems  to  be  a  question  of  the  amount  of  food.  Saito  thinks  that  the 
deprivation  of  food  is  the  main  factor  inducing  sporulation  but  that 
Schizosaccharomyces  octosporus  is  an  exception  to  this  rule. 

But  these  two  factors,  the  lack  of  food  and  the  accumulation  of 
toxic  products,  are  not  sufficient  in  themselves  to  determine  the  for- 
mation of  spores.  The  investigations  of  Hansen  and  Barker  1  have 
shown  that  there  are  a  number  of  secondary  conditions  which  are 
necessary:  free  access  of  air,  temperature,  humidity  and  condition  of 
the  cells.  More  recent  researches  by  Purvis  and  Warwick  have  shown 
that  light  exercises  an  influence  on  sporulation.  We  shall  now  take 
up  successively  these  various  conditions. 

A.  Condition  of  the  Cells.  In  order  for  a  cell  to  sporulate  it 
is  necessary  that  the  .cells  be  young  and  vigorous  and  that  they  have 
accumulated,  either  from  former  culture  media  or  from  the  medium 
in  which  they  are  taken,  a  reserve  of  products  necessary  for  the  for- 
mation of  ascospores.  We  have  seen,  indeed,  that  cells  destined  to 
form  ascospores,  have  accumulated  metachromatic  corpuscles,  fats 
and  glycogen  which  are  finally  absorbed  by  the  ascospores  during  their 
formation.  The  media  in  which  yeasts  are  placed  are  of  much  impor- 
tance in  relation  to  sporulation.  Hansen  reported  that  dextrose  had 
a  favorable  influence  on  sporulation  in  Saccharomyces  Ludwigii,  and 
Klocker  has  reported  the  same  observation  for  certain  Pichia;  these 
yeasts  only  formed  ascospores  after  a  preliminary  cultivation  in  beer 

1  Barker,  P.  On  the  spore  formation  among  the  Saccharomycetes.  Jour, 
of  the  Federated  Institutes  of  Brewing,  8,  1902. 


116  PHYSIOLOGY  OF  YEASTS   (Continued) 

wort  to  which  dextrose  had  been  added  and  others  to  which  alcohol 
had  been  added. 

The  recent  investigations  of  Saito  1  have  furnished  an  explanation 
of  some  of  these  facts  and  given  information  with  regard  to  the  for- 
mation of  ascospores  which  have  been  overlooked  up  to  the  present 
time.  Certain  yeasts  when  placed  in  an  environment  with  little  food 
do  not  contain  certain  necessary  chemical  substances  which  vary  with 
the  yeast.  For  Zygosaccharomyces  mandshuricus  which  has  been  the 
special  object  of  Saito's  investigations,  these  substances  are  dextrose, 
levulose,  galactose,  sorbose,  raffinose,  mannite,  dulcite,  sorbite  and 
glycerol.  These  substances  seem  to  exercise  a  stimulating  effect  on 
the  sporogenic  function.  There  seemed  to  be  a  mmimum  concentration 
for  each  of  these  compounds  for  sporulation.  For  example,  the  mini- 
mum concentration  of  dextrose  and  levulose  for  Zygosaccharomyces 
mandshuricus  was  between  0.125  and  0.25  per  cent;  for  galactose, 
raffinose,  and  glycerol  between  0.25  and  0.50  per  cent,  and  for  sor- 
bose and  dulcite  between  0.5  and  1  per  cent.  The  addition  of  small 
amounts  of  potassium  phosphate  and  peptone  exercised  a  favorable 
action  on  sporulation  in  Zygosaccharomyces  mandshuricus.  The  salts 
of  sodium,  potassium,  magnesium  and  carbon  had  a  favorable  action. 

Some  substances,  such  as  beer  wort  to  which  gelatin  had  been 
added  and  decoction  of  koji,  had  a  stronger  action  on  the  produc- 
tion of  ascs  than  the  carbohydrates.  Indeed,  the  action  of  beer  wort 
and  decoction  of  koji  caused  a  large  number  of  ascs,  but  among  them 
were  some  with  no  ascospores.  These  various  substances  seem,  then, 
to  have  a  specific  action  not  only  on  the  production  of  ascs  but  also 
on  the  formation  of  ascospores  and  their  maturation. 

Aside  from  substances  which  stimulate  the  formation  of  asco- 
spores, there  are  substances  which,  in  a  marked  manner,  retard  sporu- 
lation. The  salts  of  ammonia  have  such  an  action  and  when  yeasts 
are  placed  in  certain  concentrations  of  these  salts,  although  all  other 
factors  are  favorable  for  sporulation,  they  do  not  sporulate.  In  gen- 
eral it  seems  that  those  yeasts  in  which  the  asc  is  preceded  by  a  sexual 
process  sporulate  under  more  complex  conditions  than  those  which  are 
parthenogenetic. 

Sartory2  has  noticed  a  symbiosis  between  a  yeast  and  bacterium. 
The  yeast  sporulated  only  in  association  with  the  bacterium.  Zetlin  3 

1  Saito,  K.     Untersuchungen  iiber  die  chemischen  Bedingungen  fur  die  Ent- 
wicklung  der  Fortpflanzorgane  beinieigen  Hefen.    J.  College  of  Sci.  Imperial  Univ. 
Tokyo,  39,  1916. 

2  Sartory,  A.    Sporulation  d'une  levure  sous  1'influence  d'une  bacte*rie.    Comp. 
Rend.  Soc.  Biol.  72,  1912. 

3  Zetlin,   Sophie.     Influence  of  previous  nourishment  upon  spore    formation 
in  yeast.    Chemical  Abstracts,  8  (1914),  3807. 


PHYSIOLOGICAL  CONDITIONS  OF  SPORULATION     117 

studied  the  effect  of  certain  foods  on  spore  formation.  It  was  found 
that  ammonium  sulfate,  asparagin,  glycocoll  and  peptone  had  a 
favorable  action.  Spore  formation  was  greatly  increased.  The  re- 
sults with  different  sugars  were  variable.  Some  favored  spore  for- 
mation while  others  tended  to  repress  it. 

B.  Influence   of  Air.     Another  indispensable  condition  is  a  free 
access  of  air.    Hansen  has  demonstrated  this  by  the  following  example. 
Some  young  cells  of  S.  cerevisiae  and  S.  Pastorianus  are  inoculated 
into  a  Freudenreich  flask  containing  a  little  water  (about  5  drops  in 
each  flask),  and  deprived  of  air.     A  first  lot  of  these  flasks  is  placed 
into  a  bell  jar  with  a  little  alkaline  pyrogallol  and  from  which  the 
air  has  been  sucked  out  as  far-  as  possible.    Another  lot  is  placed  under 
another  bell  jar  in  contact  with  air.     Both  are  placed  at  25°  C.     After 
six  days  the  lots  should  be  examined  and  it  will  be  noticed  that  the 
yeast  cells  in  the  first  lot  will  contain  no  ascospores  while  the  cells 
in  the  second  lot  will  contain  a  large  number.     If  now  the  flasks  of 
the  first  lot  be  exposed  to  air  an  abundant  crop  of  ascospores  will  be 
noticed  after  a  few  days.     Thus  the  lack  of  air  inhibits  sporulation 
and  the  access  of  air  is  indispensable  to  the  formation  of  ascospores. 
On  the  other  hand  it  is  the  oxygen  of  the  air  which  is  so  indispensable 
to  the  formation  of  ascospores.    Hansen  has  demonstrated  this  by 
using  nitrogen  as  the  atmosphere  and  under  these  conditions  much 
less  sporulation  was  secured. 

Then,  oxygen  is  an  indispensable  factor  for  the  formation  of  asco- 
spores. Sporulation,  in  this  regard,  acts  in  a  very  different  manner 
than  budding  which,  as  we  have  stated,  is  able  to  go  on  in  the  absence 
of  oxygen. 

C.  Temperature.     One  of  the  factors  essential  to  sporulation  is 
temperature.     The   investigations   of   Hansen   have   shown   that,  for 
each  variety  of  yeast,  there  exist  certain  temperature  limits  outside 
of  which  sporulation  becomes  impossible.     Between  these  limits,  the 
time  necessary  for  the  formation  of  ascospores  is  constant  for  a  variety 
for  a  given  temperature.     Outside  of  these  temperatures,  there  are 
others  more  or  less  favorable  at  which  ascospores  form  after  varying 
lengths  of  time.    Hansen  has  shown  that  we  may  put  down  for  each 
variety: 

First.  The  temperature  limits  which  allow  the  formation  of  asco- 
spores. A  maximum  and  minimum. 

Second.  The  optimum  temperatures  at  which  ascospores  appear 
most  rapidly. 

Third.  Temperatures  which  are  between  these  limits  and  at  which 
the  time  of  ascospore  formation  is  more  or  less  long,  depending  on 
how  far  it  is  removed  from  the  optimum. 


118  PHYSIOLOGY  OF   YEASTS   (Continued) 

In  determining  these  three  temperatures  for  6  varieties  of  yeasts 
(S.  cerevisiae,  Pastorianus,  intermedius,  validus,  ellipsoideus  and  tur- 
bidans)  Hansen  has  noticed  that  sporulation  is  dependent  upon  three 
laws  which  are  able  to  be  announced  as  follows: 

First.  Sporulation  is  accomplished  slowly  at  low  temperature 
but  increases  with  the  rise  in  temperature  up  to  a  certain  optimum; 
above  this  temperature  ascospore  formation  becomes  slower  and  slower 
until  it  finally  ceases  completely.1 

Second..  The  most  favorable  temperature  for  six  varieties  of 
yeasts  was  about  25°. 

The  temperature  limits  of  these  yeasts  with  regard  to  sporulation 
are  situated  between  0.5  and  37.5°  C.  It  is  interesting  to  note  that, 
as  for  the  formation  of  the  scum,  the  temperature  limits  for  sporu- 
lation are  included  in  the  limits  of  budding.  The  minimum  tempera- 
ture for  sporulation  is  not  as  low  as  that  for  budding  and  the  maxi- 
mum is  not  so  high.  Sporulation  in  order  to  be  accomplished  requires 
a  temperature  more  pronounced  than  budding.  It  seems  that  the 
higher  the  temperature  for  budding,  the  higher  is  the  maximum  tem- 
perature for  sporulation.  The  experiments  of  Hansen  indicate,  how- 
ever, that  there  is  no  parallelism  between  the  two  temperature  curves 
for  budding  and  sporulation. 

The  observations  of  Hansen  mentioned  above,  as  well  as  those  of 
many  other  investigators  as  Nielsen,  Klocker,  and  many  others,  on 
maximum  and  minimum  temperature  limits,  have  confirmed  this. 
Nevertheless,  some  species  are  able  to  attain  maximum  temperatures 
of  40-41°.  Sometimes  they  are  situated  around  15°  C.  as  in  certain 
Pichia  studied  by  Klocker.  With  regard  to  optimum  temperatures, 
they  range  around  25°.  These  temperatures  and  the  times  required 
for  ascospore  formation  vary  with  the  yeast.  Hansen  has  been  able 
to  establish  very  important  characteristics  for  the  differentiation  of 
species. 

The  action  of  temperature  has  been  mentioned  by  Saito  in  relation 
to  formation  of  the  asc  in. certain  yeasts.  Saito  isolated  a  Zygosac- 
charomyces  which,  depending  on  the  temperature,  formed  ascs  derived 
from  a  copulation,  or  parthenogenetically.  On  slices  of  carrot  on  which 
the  yeast  germinates  very  actively,  the  ascs  were  formed  by  a  copu- 
lation at  25  to  27°  C.  At  33°-39°  C.,  on  the  contrary,  the  ascs  were 
produced  by  a  parthenogenesis. 

1  Herzog  has  shown  that  the  curves  which  show  this  phenomenon  resemble 
those  of  Tamman  on  the  influence  of  temperature  on  diastatic  action;  these 
reach  a  maximum  and  decrease  progressively.  According  to  the  same  author, 
they  also  agree  with  van't  Hofif's  law  that  the  speed  of  a  chemical  reaction  is  a 
function  of  the  temperature. 


PHYSIOLOGICAL  CONDITIONS  OF  SPORULATION     119 

D.  Humidity.     Naegeli   has   argued  that  the  principal  factor   in 
sporulation  is  desiccation  and  that  this  phenomenon  is  brought  about 
only  in  cells  which  are  partly  dried.     The  investigations  of  Hansen, 
on  the  contrary,  have  shown  that  humidity  is  an  important,  if  not 
indispensable,  condition  of  sporulation.     It  is  easy  to  show  this  by 
Hansen's  own  experiment. 

Blocks  of  plaster  of  Paris  are  prepared  and  then  plunged  into 
water  in  order  to  soak  them;  on  each  of  these,  a  little  of  the  yeast 
is  placed  and  the  blocks  placed  in  dishes  without  water,  covered  with 
a  glass  plate.  Some  other  blocks  are  placed  in  dishes  covered  with 
a  filter  paper  and  still  containing  no  water.  Another  series  of  blocks 
are  placed  in  dishes  with  water  and  covered  with  a  glass  plate.  In 
a  few  days  it  will  be  seen  that  the  yeasts  in  the  dishes  with  water 
have  formed  numerous  ascospores;  those  in  dishes  without  water  and 
covered  with  the  glass  plate  have  a  few;  those  in  the  dishes  covered 
by  a  filter  paper  have  still  less. 

The  evaporation  of  water  hindered  the  formation  of  ascospores. 
Humidity  is  then  necessary  for  sporulation.  Evaporation  does  not 
completely  stop  the  formation  of  spores,  for  a  few  cells  will  sporulate 
on  the  blocks  undergoing  evaporation.  Thus,  we  are  able  to  explain 
sporulation  in  yeasts  in  nature  on  fruits;  in  the  superficial  layers  of 
the  soil  they  are  capable  of  sporulating  in  spite  of  the  absence  of 
humidity. 

E.  Light.    According  to  the  investigations  of  Purvis  and  Warwick  1 
the  rays  of  certain  wave  lengths  have  an  influence  on  sporulation. 
By  placing  moist  plaster  of  Paris  blocks  in  dishes,  the  walls  of  which 
were  covered  with  different  colors,  they  were  able  to  establish  the 
following  facts: 

1.  The  red  rays  of  longer  wave  length  accelerate  the  formation 
of  ascospores  which  appear  more  rapidly  than  in  the  presence  of  white 
light.     They  also  seem  to  be  more  favorable  to  sporulation  than  ob- 
scurity and  seem  to  stimulate  sporulation. 

2.  The  green  rays  seem  to  retard  the  formation  of   ascospores. 

3.  The   blue   or  violet   rays   retard   sporulation  more   effectively 
than  the  green. 

4.  Finally,    the   ultra-violet   rays   have    a    pronounced    retarding 
action;    they  seem  to  have  a  bad  effect  on  the  vitality  of  the  cells. 

These  results  are  able  to  be  explained  on  a  chemical  basis,  for  it  is 
well  known  that  the  rays  of  short  wave  length  have  a  greater  chemical 
activity  than  the  long  wave  lengths.  Also  it  might  be  regarded 
that  the  former  determine  the  chemical  modifications  in  the  cell  which 

1  Purvis,  E.  and  Warwick,  R.  The  influence  of  spectral  colors  on  the  sporu- 
lation of  Saccharomyces.  Proceedings  of  the  Cambridge  Society,  14,  1907. 


120  PHYSIOLOGY  OF   YEASTS   (Continued) 

are  unfavorable  to  the  formation  of  ascospores,  while  the  rays  with 
longer  wave  lengths,  having  a  lower  chemical  energy,  have  little  in- 
fluence and  permit  the  formation  of  ascospores. 

Acid  or  Alkaline  Media:  The  investigations  of  Saito  have  shown 
that  the  degree  of  acidity  or  alkalinity  determines  sporulation  and  an 
increase  in  acidity  or  alkalinity  is  accompanied  by  a  retardation  in 
the  formation  of  ascospores.  The  higher  limits  of  acidity  for  Zygosac- 
charomyces  Mandshuricus  on  plaster  blocks  are  0.5-1.0  per  cent  of  sul- 
furic  acid,  malic  acid,  tartaric  or  citric  acid.  The  higher  limits  of 
alkalinity  are  0.2  to  0.4  per  cent  of  sodium  hydroxide.  Certain  toxic 
substances  also  affect  the  sporulation. 

Osmotic  Action:  This  also  exerts  an  effect  on  sporulation.  The 
maximum  concentration  for  spore  formation  in  a  yeast  depends  upon 
the  species.  For  an  osmophilic  species  like  Zygosaccharomyces  Mand- 
shuricus  the  concentration  is  high.  In  a  substrate  with  25  per  cent  of 
salt  this  yeast  still  sporulates.  The  investigations  of  the  action  of  other 
salts  towards  this  yeast  give  data  which  depend  upon  the  nature  of 
the  salt.  Thus,  a  very  concentrated  solution  of  potassium  nitrate 
has  much  less  effect  than  isotonic  solutions  of  NaCl.  On  the  other 
hand,  the  method  of  using  the  substance  also  determines  the  results. 

Parasitism  of  the  Yeasts.    Pathogenic  Properties.    Symbiosis 

Quite  a  number  of  the  yeasts  are  parasites.  They  seem  to  have 
received  less  attention  than  the  other  vegetable  parasites.  It  is  only 
with  animals  and  man  that  the  parasitism  in  the  yeasts  is  of  any  im- 
portance. Endomyces  albicans,  a  fungus  related  to  the  yeasts,  has 
been  known  for  a  long  time  to  cause  lesions  in  man.  Remack  has  found 
in  the  intestines  of  mammals  a  true  yeast  capable  of  sporulation  which 
received  the  name  of  Saccharomycopsis  guttulatus.  Metschnikoff  in 
1884  discovered  in  the  general  cavity  of  a  crustacean  (Daphnia)  a  yeast 
which  caused  a  special  infection.  It  was  by  means  of  this  yeast,  on 
account  of  the  thin  wall  of  the  daphnia,  that  Metschnikoff  discovered 
phagocytosis.  The  yeast  was  called  Monospora  cuspidata. 

Other  pathogenic  yeasts  have  been  observed  in  other  animals. 
Biitschli  and  Dangeard  have  found  them  in  the  Anguillula,  Schaudin 
in  the  intestines  of  Culex  and  Lindner  in  the  larvae  of  the  fly  Core- 
thra  plumicorum. 

For  a  dozen  years  the  number  of  pathogenic  yeasts  has  been 
notably  increased  by  the  discovery  of  different  ones  which  cause 
various  troubles  in  man  and  animals  under  the  name  of  blastomyco- 
sis.  Rivolts  and  Micellone  have  described  the  Cryptococcus  farcimi- 
nosus  which  causes  farcy  in  the  horse.  Raynaurd,  Lucet  and  Guegen 


PATHOGENIC   PROPERTIES  OF  YEASTS  121 

have  described  the  Cr.  linguae-pilosae  which  causes  a  malady  in  man. 
Achalme  and  Troissier  found  S.  anguinae  which  caused  an  angina. 
According  to  Le  Dantec  certain  dysenteries  may  be  caused  by  yeasts. 
Quite  a  number  of  yeasts  have  been  described  in  tumors.  One  of  the 
most  characteristic  of  these  is  the  yeast  of  Curtis,  Saccharomyces 
subcutaneous  tumefaciens,  which  is  a  true  yeast  forming  ascospores. 
Blanchard,  Schwartz  and  Binot  have  described  a  yeast  which  caused 
a  tumor.  Vuillemin  and  Legrain  have  isolated  S.  granulatus  which 
had  pathogenic  properties. 

The  frequency  of  pathogenic  yeasts  has  lead  certain  authors  to 
attribute  to  these  fungi  a  varied  role  in  disease,  especially  for  some 
of  those  diseases  for  which  bacteria  have  not  been  discovered.  Thus 
attempts  have  been  made  to  explain  rabies  and  cancer  on  the  basis 
of  the  presence  of  yeasts.  The  possible  relation  of  yeasts  to  cancer 
has  held  the  attention  of  bacteriologists  and  a  brief  resume  of  the 
subject  will  be  presented  although  the  subject  has  been  abandoned 
today  and  is  of  historical  interest  only.  The  presence  of  yeasts  (Cur- 
tis, Blanchard,  Schwartz  and  Binot,  Vuillemin  1  and  Legrain)  in  many 
tumors  has  suggested  that  possibly  these  organisms  were  the  cause. 

This  idea  has  been  supported  especially  by  Russel  who  observed,  in 
a  large  number  of  carcinomas,  spherical  bodies  which  he  called  yeasts. 
The  investigations  of  Corselliet,  Frisco,  Plimmer,  and  Bra  seem,  at  first 
thought,  to  confirm  this  opinion.  These  investigators  isolated  a  yeast 
from  many  tumors  (sarcomas,  epitheliomas  and  carcinomas).  Plim- 
mer especially  found  Cr.  Plimmer i  in  more  than  one  thousand  carci- 
nomas. On  the  other  hand  San  Felice  pretended  to  have  provoked 
the  formation  of  true  neoplasms  by  animal  inoculation  of  a  yeast 
isolated  from  certain  fruits,  the  Cr.  neoformans.  For  a  time  this  patho- 
genic theory  of  yeasts  for  cancer  held  a  very  strong  position  among 
clinicians  and  anatomo-pathologists. 

It  is  generally  agreed  that  among  all  of  the  published  observations, 
there  is  not  one  which  will  stand  close  scrutiny  and  which  is  suffi- 
ciently demonstrative.  It  is  now  known  that  the  bodies  which  Russel 
observed  are  only  degenerate  cytoplasm.  On  the  other  hand,  Ron- 
cali,  Plimmer  and  Bra  have  run  afoul  in  their  animal  inoculation  ex- 
periments, for  the  yeasts  which  were  isolated  never  reproduced  the 
tumors. 

If  certain  investigators,  among  others  Carselli  and  Fisco,  San 
Felice,  have  been  able  to  produce  true  tumors  by  inoculation  of 
yeasts,  it  was  never  demonstrated  that  the  tumors  had  any  histolog- 

1  Vuillemin,  P.  Cancer  et  tumeurs  ve"getales,  Bull,  des  seances  de  la  Soc. 
des  Sc.  de  Nancy,  1900.  Les  Blastomycetes.  Rev.  g£n.  des  Sciences,  1901, 
No.  16. 


122  PHYSIOLOGY  OF  YEASTS   (Continued) 

ical  similarities  to  cancer.  The  inoculation  and  rapid  increase  in  loco 
of  these  yeasts  alone  determine  the  common  lesions  exactly  as  in  all 
foreign  bodies.  According  to  Maffuci  and  Sirleo,  many  yeasts,  which 
have  been  isolated  from  malignant  tumors,  came  from  the  air  and 
not  from  the  diseased  tissue.  These  authors  have  been  able  to  isolate 
yeasts  from  numerous  carcinomas  and  sarcomas,1  but  they  also  ob- 
tained them  by  exposing  gelatin  plates  to  the  air  of  the  laboratory. 

It  then  seems  probable  that  many  of  the  yeasts,  said  to  have  been 
isolated  from  cancer  tissue,  really  came  from  the  air.2 

It  seems  certain,  however,  that  yeasts  may  be  found  in  certain 
malignant  tumors,  but  they  must  be  of  only  secondary  importance  in 
the  disease.  They  never  cause  it.  The  yeasts  develop  simply  be- 
cause they  find  the  organism  weak  and  in  the  neoplastic  tissue  a 
favorable  environment  —  a  good  culture  medium.  One  is  able,  for 
example,  to  secure  Endomyces  albicans  in  certain  tumors  which,  as 
is  generally  known,  causes  an  infection  in  infants,  aged  and  gener- 
ally "run-down "  individuals.  The  blastomycelial  theory  of  cancer 
has  been  definitely  rejected. 

Pathogenic  Properties  of  Yeasts 

Even  though  it  is  admitted  today  that  yeasts  have  no  relation 
to  cancer,  it  is  possible  to  inquire  whether  certain  yeasts,  either 
special  yeasts  or  common  saprophytes,  are  not  capable  of  present- 
ing, at  times,  toxic  or  pathogenic  properties.  Generally  speaking, 
the  question  has  been  answered  in  the  negative,  and  it  is  now  rec- 
ognized that  the  yeasts  are  almost  without  significance  for  the  higher 
animals. 

Rabinowitch,3  in  inoculating  50  varieties  of  ordinary  yeasts  into 
various  animals,  found  only  seven  which  were  pathogenic  for  the 
mouse  and  rabbit.  None  caused  even  the  slightest  reaction  in  the 
guinea  pig.  The  animals  which  were  killed  seemed  to  be  dead  from 
infection  and  not  intoxication.  Yeasts  do  not  seem  to  secrete  a  toxin 
which  has  any  action  on  animals. 

The  results  of  investigations  conducted  by  Skchiwan  4  have  shown 
that  yeasts  have  practically  no  chemiatric  action  towards  leucocytes. 
This  was  demonstrated  by  introducing  S.  pastorianus  in  capillary 
tubes  into  the  peritoneum  of  guinea  pigs  and  rabbits. 

1  Guegen,  F.    Les  Champignons  Parasites  de  I'homme  et  des  animaux.    These 
d'agre*g.  de  Pharmacie,  Paris,  1902. 

2  Guiart,  J.    Precis  de  parasitologie.    Baillierie  edit.  Paris,  1910. 

3  Rabinowitch,    L.     Untersuchungen   iiber   pathogene   Hefearten.     Zeitschr. 
Hyg.  21,  1896. 

4  Skchiwan.     Ann.  Inst.  Past.  8,  1890. 


PATHOGENIC   PROPERTIES   OF   YEASTS  123 

By  injecting  into  the  peritoneal  cavity  of  a  guinea  pig  a  culture 
of  S.  pastorianus  and  examining  what  happened,  by  removing  a  little 
of  the  peritoneal  fluid  at  regular  intervals,  the  same  author,1  observed 
a  phagocytosis  of  the  yeasts.  They  were  demonstrated  to  be  alive,  how- 
ever, by  means  of  inoculation.  After  from  2  to  3  hours  they  were 
broken  up  in  the  interior  of  the  leucocytes  and  at  the  end  of  from  10 
to  11  days  they  were  proven  to  be  dead  by  means  of  inoculations  into 
beer  wort.  If,  however,  in  place  of  an  ordinary  yeast,  a  pathogenic 
yeast  be  used,  such  as  S.  subcutaneous  tumefaciens,  the  same  phe- 
nomena are  observed  with  the  single  difference  that  phagocytosis 
is  more  energetic.  This  commences  after  a  sort  of  lag,  at  first  by  the 
polynuclear  leucocytes  and  finally  the  mononuclears;  often  a  cell  is 
observed  which  has  been  ingested  by  many  leucocytes.  On  the  other 
hand,  a  yeast  may  defend  itself  by  surrounding  itself  by  a  mucilag- 
inous envelope.  A  battle  between  the  yeasts  and  the  leucocytes 
ensues.  But  finally  the  leucocytes  triumph  and  at  the  end  of  two  or 
three  days  all  the  yeast  cells  find  themselves  ingested. 

It  may  be  inferred,  then,  that  ordinary  yeasts  do  not  exhibit  a 
pathogenic  phase  and  that  pathogenic  yeasts  themselves  provoke 
only  a  light  of  doubtful  intoxication.  According  to  Casagrandi,2 
and  Demme 3  a  yeast,  Crypt,  ruber,  caused  an  acute  enteritis  in  young 
infants.  The  active  agent  was  probably  a  secondary  cleavage  prod- 
uct from  the  milk  in  which  it  was  secured. 

If  one  summarizes  the  numerous  observations  of  secondary  in- 
fections by  yeasts  and  the  diverse  lesions  of  the  skin,  mucous  mem- 
branes or  internal  organs,  the  cases  are  rare  where  these  fungi  exhibit 
any  actual  pathogenic  r61e.  Blastomycoses  thus  seem  to  be  excep- 
tional diseases  and  not  so  very  frequent.4 

That  we  drink  with  wine  large  numbers  of  yeasts  might  indicate 
their  harmlessness.  For  a  long  time  we  have  attributed  curative  prop- 
erties to  beer  yeasts  toward  such  infections  as  furunculosis.  Ser- 
gent 5  has  noticed  an  antiseptic  action  of  yeasts  against  infections  with 
Staph.  pyogenes  aureus.  Perhaps  these  properties  find  their  explana- 
tion in  the  existence  of  a  toxin  recently  demonstrated  by  Hayduck, 
Fernbach  and  Vulqium.  It  has  been  shown  in  the  preceding  chapter 
that,  according  to  certain  authors,  yeasts  secrete  a  toxin  endowed 

1  Skchiwan,  Ann.  Past.  Inst.  13,  1899. 

2  Casagrandi,    O.    Saccharomyces   ruber.    Ann.    d'Ig.   sperm.    1898.    Vok.   7 
and  8. 

3  Demme,  R.    Saccharomyces  ruber.     Ann.  de  microg.  1889  and  Annali  d'Ig. 
sperm.  1897,  Vol.  7. 

4  Duval  and  Laederich.    Arch,  de  Protistology  3. 

6  Sergent,  E.     Levure  de  biere  et  suppuration     Ann.  Inst.  Past.  17,  1903. 


124  PHYSIOLOGY   OF    YEASTS    (Continued) 

with  a  decided  bactericidal  power.1  The  investigations  of  Neumayei 
and  Anderson  seem  to  indicate  that  the  yeasts  are  able  to  withstand 
the  action  of  the  digestive  juices  and  may  thus  pass  through  the  di- 
gestive canal.  Hawk  and  his  colleagues  at  Jefferson  Medical  College 
have  reported  on  the  value  of  yeasts  in  the  treatment  of  furunculosis. 
They  claimed  better  results  than  were  secured  by  the  use  of  autog- 
enous vaccines.  The  use  of  yeasts  in  therapeutics  is  not  a  new  idea. 
In  the  earliest  of  times  they  were  used  against  the  pyogenic  cocci. 

Symbiosis  of  Yeasts 

Symbiosis  is  the  association  of  two  different  organisms  which  live 
together,  both  being  benefited.  It  seems  that  yeasts  are  able  to  un- 
dergo similar  associations. 

Thus  it  is  that  in  certain  industrial  yeasts  made  up  of  differ- 
ent fermenting  agents  there  is  a  living  together  or  a  symbiosis.2 
Will  has  reported  the  case  of  two  varieties  of  yeast  which  have  func- 
tioned in  a  brewery  for  12  years  without  any  noticeable  change  in 
their  individual  characteristics.  A  sort  of  equilibrium  seems  to  have 
been  established  between  the  two  varieties  which  permits  them  to 
live  together  without  harming  each  other.  Schonfeld  has  cited  a 
similar  case  of  a  little  brewery  in  which  the  leaven  for  four  years 
gave  a  rapid  clarification  with  feeble  alternation.  This  leaven  was 
composed  of  two  yeasts,  one  which  had  low  alternation  and  the  other 
with  higher  alternation.  These  two  species  lived  for  two  years  in 
close  contact  without  harm  and  preserved  their  relative  strengths. 
Van  Laer  has  also  noticed  a  case  of  equilibrium  among  yeasts  in  the 
innoculum  of  a  top  fermentation  and  which  for  a  long  time  lived  in 
symbiotic  relations.  In  this,  two  yeasts  predominated;  first,  a  yeast 
of  the  type  c&revisiae  which  caused  saccharose  and  maltose  to  fer- 
ment; secondly,  a  Torula  A  which  caused  saccharose  to  ferment, 
but  which  acted  on  maltose  a  little  and  which  gave  the  beer  an  agree- 
able taste  and  odor.  Two  other  varieties  were  present  to  a  lesser 

1  The  use  of  yeasts  in  nutrition   has  received  some  attention.    Voelz   and 
Baudrexel   (Ann.  de  la  Brasserie  et   de  la  Distillerie,   1911)   have  shown,  by  a 
series  of  experiments  with  dogs,  that  yeasts  constitute  a  good  source  of  nitrogen. 
At   the  suggestion  of  Professor  Delbruck,  a  dozen  assistants  at  the  Fermentation 
Institute  at   Berlin   have   replaced,   for  several   weeks,   a  part   of  their  meat  at 
breakfast  by  20  gms.  of  dried  yeast.     None  of  them  suffered  any  trouble  by  the 
introduction  of  this  new  food  (Delbruck,  La  Levure,  un  noble  champignon,  1st 
International  Congress  of  Brewing,   Brussels,    1910).     Voltz   (Biochem.   Zeit,   93, 
101-5)  has  stated  that  yeast  should  not  be  fed  in  the  living  condition  if  it  is  to 
be  of  food  value. 

2  These  examples  are  taken  from  Duclaux,  Traite  de  microbiologie, 


SYMBIOSIS   OF  YEASTS  125 

proportion,  two  yeasts  of  the  Pastorianus  type;  one  A,  was  rather  in- 
active, the  other  B  seemed  to  take  part  in  the  secondary  fermentation. 
But  it  is  necessary  to  say  that  such  associations  in  yeasts  xare  rare. 
In  most  of  the  breweries  where  mixtures  of  yeasts  are  employed,  it 
is  exceptional  that  an  equilibrium  is  maintained  between  them.  One 
almost  always  predominates  over  the  others. 

Certain  fermentations  in  distilleries  produced  by  mixtures  of 
fungi  also  seem  to  be  cases  of  symbiosis.  The  fungi  transform  the 
starch  into  maltose  which  the  yeasts  ferment.  A  large  number  of  fer- 
mented beverages  used  in  different  countries,  resulting  from  the  fer- 
mentation of  starch,  seem  to  be  due  to  such  symbiotic  associations  of 
yeasts  and  fungi.  For  example,  Sake,  an  alcoholic,  drink  prepared 
by  the  fermentation  of  rice,  and  used  in  Japan,  is  an  example.  This 
beverage  is  obtained  by  the  action  of  different  organisms,  among  which 
is  Aspergillus  oryzae  and  many  yeasts  and  molds.  The  starch  of  the 
rice  is  changed  into  maltose  by  Aspergillus  oryzae  and  this  sugar  fer- 
mented by  the  molds  and  yeasts.  Arrack,  obtained  by  the  fermentation 
of  molasses  and  rice  flour,  is  produced  by  an  agent  made  up  of  bac- 
teria, molds  (Chlamydomucor  oryzae,  Rhyzopus  oryzae)  and  two  yeasts. 
The  fermentation  in  bread  seems  to  result  by  the  symbiotic  action  of 
a  yeast  and  bacteria. 

Another  example  of  the  same  order  has  been  mentioned  by  Lutz.1 
According  to  this  author,  tiby,  an  alcoholic  drink  of  the  Mexicans, 
is  produced  by  the  action  of  a  yeast,  Pichia  Radaisia,  and  a  bac- 
terium B.  mexicanus,  which  live  in  symbiotic  association.  The  yeast 
living  in  contact  with  air  is  not  able  to  induce  fermentation.  Asso- 
ciated with  the  bacillus  it  brings  about  the  alcoholic  fermentation.  The 
bacterium  plays  its  only  role  in  keeping  the  concentration  of  oxygen 
down.  Lutz  has  been  able  to  bring  about  an  experimental  symbiosis 
with  P.  Radaisia  and  B.  subtilis. 

Another  classic  example  of  symbiosis  has  been  observed  by  Freu- 
denreich 2  in  kefir,  the  fermented  milk.  He  has  isolated  4  micro- 
organisms: 1,  the  kefir  yeast;  2,  Streptococcus  a  which  coagulates 
the  milk;  3,  Streptococcus  b  which  possesses  probably  a  lactase;  and 
4,  Dispora  caucasica  or  Bacillus  caucasicus  whose  role  is  not  known. 
According  to  Freudenreich,  the  yeast  does  not  possess  a  lactase  and  is 
thus  unable  to  ferment  lactose.  This  is  accomplished  by  Streptococ- 
cus b.  This  is,  then,  a  symbiotic  association.  Jorgensen  has  observed 
another  yeast  in  kefir,  S.  fragilis,  which  possesses  a  lactase  and  is  thus 

1  Lutz,  L.    Association  symbiotique  du  Sacch.  Radaisii.    Bull,  de  la  soc.  myc. 
de  France,  1906. 

2  Freudenreich,  E.     Bakteriologische  Untersuchungen  uber  den  Kefir.     Cent. 
Bakt.  3,  1897. 


126  PHYSIOLOGY  OF   YEASTS   (Continued) 

able  to  ferment  lactose.  Beijerinck  has  also  confirmed  this.  This 
author  found  S.  Kephir. 

Sartory l  has  recently  described  a  yeast  which  sporulated  only 
in  the  presence  of  a  bacterium  with  which  it  lives. 

Rist  and  Khoury,2  on  the  other  hand,  have  observed  a  similar 
phenomenon  in  the  fermentation  of  leben,  an  Egyptian  fermented 
milk.  This  fermentation  is  due  to  two  yeasts,  the  S.  lebenis  and 
Mycoderma  lebenis,  and  a  bacterium  Streptococcus  lebenis.  The  two 
yeasts  alone  are  not  able  to  ferment  the  lactose.  But,  associated  with 
Streptococcus  lebenis,  they  bring  about  the  fermentation  of  the  milk. 
The  bacterium  seems,  then,  to  act  on  the  lactose  in  some  way  to  make 
it  available  for  .the  yeasts. 

Another  very  curious  example  of  symbiosis  is  furnished  by  the 
parasites  in  that  rare  infection  in  man  known  as  "  black  tongue." 
Guegen's  3  work  seems  to  indicate  that  this  disease  is  caused  by  a 
mold  Oospora  lingualis  and  a  yeast  Cryptococcus  linguae-pilosae.  The 
yeast  functions  only  when  it  is  in  association  with  the  mold.  These 
observations  have  been  confirmed  by  the  investigations  of  Thaon.4 

Carpano 5  has  reported  that,  in  infections  by  Cryptococcus  farcimi- 
nosus,  Staph.  pyogenes  aureus  and  Strept.  adenitis  equi  are  found.  Pos- 
sibly this  is  another  symbiotic  relationship. 

It  may  be  possible  to  have  symbiotic  relations  between  certain 
yeasts  and  the  cholera  vibrio,  for  Metschnikoff  has  shown  that  this 
latter  organism  is  favored  by  the  presence  of  a  Torula. 

One  is  able  to  cite  many  examples  in  which  two  forms  of  life  are 
able  to  live  together.  However,  up  to  the  present  time,  we  have  only 
observed  symbioses  between  two  yeasts  or  between  a  yeast  and  a  bac- 
terium. It  is  possible  to  have  symbiosis  between  two  totally  dif- 
ferent organisms,  such  as  a  yeast  and  an  animal.  Thus,  Lindner 6 
found  Saccharomyces  apiculatus  parasiticus  in  an  hemoptera,  Aspidi- 
otus  Nerii,  in  which  it  was  always  present  without  seeming  to  exer- 

1  Sartory,  A.    Speculation  d'une  levure  sous  I'influence  d'une  bacterie.    Comp. 
Rend,  des  Soc.  Biol.  72,  1912. 

2  Hist,  E.  and  Khoury,  J.     Etude  sur  un  lait  fermente*   comestible.     Cent. 
Bakt.  and  Leben  d'Egypte.    Ann.  Past.  Inst.  1902,  16. 

3  Guegen,   F.     Sur   Oospora  lingualis  et   Cryptococcus   lingualis.     Arch,   de 
Parasit.  1909,  12. 

4  Thaon,  P.     Symbiose  de  levure  et  Oospora  dans  un  cas  de  langue  noire. 
Soc.  de  Biologie,  67.     1909. 

6  Carpano,  M.  The  association  of  bacteria  in  Cryptococcus  farciminosus  in- 
fection. Ann.  Ig  (Rome)  28,  273-279.  1918. 

6  Lindner,  P.  Das  Vorkommen  der  parasitischen  Apiculatus  Hefe  in  auf 
Hefen  schmarotzenden  Schildlausen  und  deren  mutmassliche  Bedeutung  der 
Nonneraupe.  Wochen.  f.  Brauerei,  No.  3,  1907, 


SYMBIOSIS   OF   YEASTS  127 

else  any  pathogenic  role.  Conte  and  Faucheron  1  nave  aiso  observed 
yeasts  in  the  fatty  tissue  of  female  coccidia.  They  were  led  to  regard 
this  observation  as  a  sort  of  symbiosis. 

This  hypothesis  has  been  confirmed  by  the  investigations  of  Pier- 
antoni  and  Karl  Sulc  which,  on  account  of  their  great  biological  in- 
terest, are  important  enough  to  mention  here.  All  of  the  authors 
who  have  studied  the  adipose  tissue  of  the  homoptera  have  noticed 
the  existence  in  these  tissues  of  special  organs  which  have  received 
different  names,  depending  on  the  insects  in  which  they  have  been 
observed,  but  which  seem  to  have  a  certain  simi- 
larity; such  are  the  pseudo-vitellius  and  green 
bodies  described  by  Huxley,  Lubhock,  Balbiani 
and  Henneguy  in  various  Aphides,  the  polar  mass 

observed  by  Heymons  in  the  eggs  of  the  Cicadides  ^.      53  A    Larva 

and  the  oval  body  found  by  Berlese  in  the  genus      of    Ptyelus  '  limatus 

Dactylopius.     The  significance  of  these  organs  has       ^  mycetome  M. 

.       .  MI  B,  Section  of  myce- 

remamed  unknown  until  the  present  time.     How-      tome  of  Ptyelus  line- 

ever  it  has  been  stated  that  they  were  cells  with      <^*     (after    Karel 

contents  of  fat  droplets  or  protein  grains.     Some 

have  stated  that  they  served  to  control  the  reserve  products. 

The  work  of  Pierantoni  and  Sulc,  carried  out  independently  at 
the  same  time,  have  shown  that  these  organs,  which  appear  to  be  in 
all  of  the  homoptera,  present  the  same  structure  and  may  be  homol- 
ogous. 

These  organs  to  the  number  of  two  in  each  insect  are  situated  on 
each  side  of  the  intestines  of  the  insect  quite  near  the  reproductive 
organs  (Fig.  53,  A).  They  are  made  up  of  a  mass  of  large  cells  with 
a  yellow  or  greenish  epithelial  membrane.  They  contain  an  ameboid 
nucleus  and  a  cytoplasm  filled  with  small  spherical  or  oval  bodies. 
These  bodies,  regarded  by  some  as  reserve  products  (fat  or  proteins), 
in  reality  resemble  the  yeasts.  These  yeasts  vary  from  one  insect  to 
another.  Budding  yeasts  are  especially  found,  among  which  is  a 
variety  already  mentioned,  Saccharomyces  apiculatus  parasiticus, 
Schizosaccharomyces  and  some  yeasts  related  to  these  latter  to  which 
Sulc  gave  the  name  of  Cicadomyces. 

These  yeasts  agree  with  those  described  by  Lindner,  Conte  and 
Faucheron  in  the  Coccidia  and  are  encountered  constantly,  probably 
being  handed  down  in  the  egg.  The  cells  situated  in  the  center  of 
these  organs  divide  regularly,  and  cause  by  their  growth  a  shattering 
of  the  superficial  layers  which  liberates  the  yeasts.  These  happen  to 
come  in  contact  with  the  ovaries  and  penetrate  the  egg  during  the 

1  Conte,  A.  and  Faucheron,  L.  Presence  de  levures  dans  le  corps  adipeux 
de  divers  Coccides.  Comp.  Rend.  Acad.  des  Sciences,  144,  1907. 


128  PHYSIOLOGY  OF  YEASTS   (Continued) 

process  of  its  formation.  They  form  a  little  mass  at  one  of  the  poles 
made  up  of  a  few  cells.  This  mass  is  soon  surrounded  by  a  number  of 
cells  resulting  from  the  segmentation  of  the  reproductive  vesicles 
and  of  blastodermic  origin.  This  polar  mass  remains  distinct  from  all 
of  the  other  organs  during  the  development  of  the  embryo.  It  is 
placed  in  the  rear  part  of  the  abdomen  of  the  growing  cells,  the  yeasts 
penetrating  into  their  interior  until  the  mass  parts  into  two  bodies 
which  take  positions  on  both  sides  of  the  intestines  as  described  above. 

Sulc  and  Pierantoni  have  been  then  led  to  conclude  that  the 
pseudo-vitellius,  green  body  or  oval  body  constitutes  organs  resulting 
from  a  sort  of  inflammation  produced  by  these  yeasts  on  the  blasto- 
dermic cells  in  the  course  of  segmentation.  Sulc  has  designated  them 
as  mycetomes,  reserving  the  name  mycetocytes  for  the  same  cells  filled 
with  yeasts. 

Both  of  these  investigators  admit  that  the  yeast  found  in  the 
mycetomes  is  living  in  symbiotic  relation  with  the  insect.  At  first 
the  yeast  was  probably  a  parasite  but  a  continued  association  with  the 
insect  brought  about  a  symbiotic  association.  According  to  Sulc  the 
mycetomes  play  a  similar  role  to  that  of  the  lymphatic  ganglions. 
They  protect  the  insect  from  the  invasion  of  bacteria,  by  means  of 
the  yeasts  which  are  contained  in  their  interiors,  for  it  has  been  shown 
by  the  investigations  of  Haydruck  and  Fernbach  that  they  are  germi- 
cidal.  On  the  contrary,  Pierantoni x  believes  that  the  mycetomes  play 
a  role  in  digestion.  The  homoptera  live  on  vegetable  sap  and  take  in 
much  starch  and  sugar.  Part  of  these  hydrocarbons  is  assimilated, 
the  rest  remaining  in  the  intestinal  tract  to  be  finally  eliminated.  This 
author  thinks  that  the  yeasts  in  the  mycetomes  serve  in  the  assimila- 
tion of  the  carbohydrates  and  in  the  transformation  to  carbon  diox- 
ide and  alcohol.  This  question  apparently  needs  further  study. 

Vandevelde 2  has  paid  considerable  attention  to  the  question  of 
symbiosis  in  yeast.  He  has  carried  rather  extensive  investigations. 
When  certain  yeasts  fermented  together,  the  fermentation  was  car-* 
ried  on  to  a  further  degree.  The  conclusions  which  this  author  draws 
from  his  investigations  are  that  mixed  yeasts  give  better  results  than 
pure  cultures  in  the  fermentation  industries.  It  is  interesting  to 

1  Pierantoni,   U.     L'origin  di  alcuni  organi   d'Icerza  perchasi  e  la  simbiosi 
eredaria.     Bull.   Soc.    Napoli,   23,    1909;     Origine    e    struttura    del    corpo    ovale 
del   Bactylopius   citri  e  del   corpo  verde   dell'  Aphis   brassicae.     Ibid.  24,   1910. 
Ulteriori    osservazioni    sulla    simbiosiereditaria    degli    omotteri.      Zool.    Auz.    36, 
1910. 

2  Vandevelde,   A.   J.   J.     Chemical   phenomena  in  the   symbiosis   of   yeasts. 
Rev.  Gen.  Chim.  18  (1915),  88-95;    On  symbiotic  life  of  yeast  races.     Original 
Communications,  8th   International   Congress  of  Applied   Chemistry,   14    (1912) 
191-202. 


SYMBIOSIS   OF  YEASTS  129 

wonder  whether  symbiosis  in  yeasts  may  not  be  explained  in  part  on 
the  basis  of  vitamines,  as  has  been  suggested  by  certain  pieces  of 
research  for  the  bacteria. 

Vitamines  in  Yeast :  The  importance  of  "accessory  substances," 
the  so-called  "  vitamines,"  in  the  treatment  of  certain  deficiency 
diseases,  has  caused  investigators  to  examine  different  substances  for 
them.  While  our  knowledge,  with  regard  to  vitamines,  seems  to  be 
in  a  transitory  state,  it  may  be  advisable  to  mention  a  few  of  the 
more  important  papers  on  the  presence  of  them  in  yeasts.  Funk  l 
isolated  2.5  grains  of  vitamine  fraction  from  100  kg.  of  dried  yeast. 
When  this  was  injected  in  the  muscle  of  a  pigeon  suffering  from  poly- 
neuritis,  complete  recovery  followed.  This  original  substance  was  fur- 
ther fractionated.  Some  of  these  were  thought,  at  that  time,  to  be 
nicotinic  acid.  Seidell 2  found  that  brewers'  yeast  was  the  cheapest  as 
well  as  the  richest  source  of  vitamines.  The  yeast  cells  are  dried  hy- 
draulically  and  allowed  to  autolyze  at  37.5°  C.  for  48  hours.  After 
cooling,  the  liquid  is  filtered.  In  this  clear  filtrate  will  be  found  about 
50  per  cent  of  the  raw  material,  and  23  per  cent  of  the  total  solids. 
One  cc.  of  this  injected  into  a  paralyzed  pigeon  caused  relief  in  an 
hour  and  a  return  to  normal  condition  in  12  hours.  Seidell 3  stated 
later  that  the  autolytic  process  influenced  the  power  of  the  vitamine. 
Emmett  and  McKim  4  found  that  the  yeast  vitamine  of  Seidell  should 
be  accompanied  by  vitamine  containing  foods  in  order  to  accomplish 
normal  gains  in  weight  and  complete  recovery.  Hawk  5  and  his  col- 
leagues, in  another  connection,  have  found  that  yeasts  are  decidedly 
beneficial  for  treating  skin  diseases.  Improved  conditions  resulted 
in  many  cases  from  ingestion  of  yeast,  where  autogenous  vaccines 
caused  no  relief.  In  many  cases  there  was  a  general  improvement  in 
the  condition  of  the  patient  "  quite  unassociated  .  .  .  with  the  partic- 
ular disease  in  question."  This  might  indicate  that  some  essential 
or  beneficial  substance  was  added  to  the  diet  through  the  ingestion 
of  the  yeast. 

That  vitamines  may  be  necessary  for  the  development  of  yeast, 

1  Funk    C.     Studies  on  Beri-beri.     Further  facts  concerning  the  chemistry 
of  the  vitamine  fraction  from  yeast.     Brit.  Med.  J.  1913,  I,  814.    J.  Physiol.  46, 
173-9. 

2  Seidell,   A.     Vitamines   and   nutritional   diseases.     Public  Health  Reports, 
31,  364-70,  1916. 

3  Seidell,  A.     The  vitaminic  content  of  brewers'  yeast.     Jour.   Biol.   Chem. 
29,  145-54,  1917. 

4  Emmett,  A.  D.  and  McKim,  L.  H.    The  value  of  the  yeast  vitamine  frac- 
tion as  a  supplement  to  a  rice  diet.    J.  Biol.  Chem.  32,  409-19,  1917. 

5  Hawk,  P.  B.  et  al.    The  use  of  bakers'  yeast  in  the  diseases  of  the  skin  and 
of  the  gastrointestinal  tract.    Jour.  Amer.  Med,  Assn.  69  (1917),  1243-1247. 


130  PHYSIOLOGY   OF   YEASTS    (Continued) 

seems  to  be  indicated  by  the  investigations  of  Williams  l  and  Bach- 
mann.2  Williams  presents  data  to  show  that  water-soluble  vitamine 
may  be  necessary  for  the  growth  of  yeasts  themselves.  Williams  used 
the  development  of  a  single  cell  of  yeasts  as  the  indication  of  the 
presence  or  absence  of  vitamine.  Substances  which  were  known  to 
be  rich  in  vitamine  were  also  found  to  be  rich  in  the  yeast  growth-pro- 
moting substance.  Williams  believes  that  these  substances  which 
stimulate  yeast  development  are  the  same  as  those  which  prevent 
beri-beri.  In  a  synthetic  solution  alone,  a  cell  of  yeast  developed  very 
slowly.  Under  identically  the  same  conditions,  with  the  exception  of 
the  addition  of  one  part  in  60,000  of  growth-promoting  substance, 
many  more  cells  were  formed  from  the  single  cell.  Bachmann  found 
that  water-soluble  B  vitamine  was  quite  necessary  for  vigorous  de- 
velopment of  a  yeast  isolated  from  canned  pears.  Both  papers  bear 
out  the  contention  of  Wildier,3  an  earlier  worker,  who  found  that  some 
substance,  to  which  he  gave  the  name  "  bios,"  was  necessary  for  vig- 
orous development  of  yeasts. 

1  Williams,   R.   J.    1919.     The  Vitamine  Requirement  of  Yeast.     A  simple 
biological  test  for  vitamines.    J.  Biol.  Chem.  38  (1919),  465-86. 

2  Bachmann,    F.    M.      Vitamine   Requirements   of   certain   yeasts.      J.    Biol. 
Chem.  39,  235-58. 

3  Wildier,  E.     Nouvelle  substance  indespensible  au  developpement  de  la  levure. 
La  Cellule,  18  (1901)  313. 


CHAPTER   V 

ORIGIN  OF  THE  YEASTS,  THEIR  POSITION  IN  CLASSI- 
FICATION  OF  THE  FUNGI  AND   THEIR 
SYSTEMATIC  RELATIONSHIPS 

LET  us  now  consider  the  morphological,  cytological,  and   physi- 
ological characteristics  of  the  yeasts.     It  is  interesting  to  con- 
sider the  place  which  they  hold  in  the  classification  of  the  fungi. 
It  has  been  stated,  at  the  beginning  of  this  book,  that  the  sporangium 
of  the  yeasts  is  comparable  to  the  asc  in  the  Ascomycetes.    It  now  re- 
mains for  us  to  discuss  the  reasons  for  wishing  to  incorporate  the 
yeasts  under  the  Ascomycetes.     Although  this  is  definitely  settled  to- 
day, this  question  has  been  the  object  of  such  polemics  that  they  are 
worthy  of  our  attention. 

(A)   Historical 

The  question  of  the  position  of  the  yeasts  in  classification  of  the 
fungi  has  remained  unsolved  for  quite  a  period  of  time.  Do  the  yeasts 
make  up  an  autonomous  species  or  do  they  simply  represent  a  stage 
in  the  development  of  the  filamentous  fungi,  more  advanced,  which 
exist  during  the  fruit  season  as  yeasts,  and  during  the  winter  as  myce- 
lial  fungi?  It  is  easy  to  observe  the  different  stages  in  the  life  history 
of  yeasts,  the  stages  of  budding  and  sporulation;  but  it  has  riot  been 
shown  that  the  culture  in  artificial  media  presents  the  whole  life  cycle 
and  that  it  may  not  be  more  complex  in  nature.  Thus,  we  have  seen 
in  the  early  part  of  this  book  that  many  fungi  present  yeast-like 
structures  during  some  stage  in  their  life  cycles.  Such  is  the  question 
that  arose  in  the  days  when  Pasteur  worked  and  which  ought  to  be 
answered  in  our  day. 

The  subject  is  rendered  more  complex  by  the  fact  that  little  is 
known  about  their  origin  and  life  cycles.  It  is  known  that  beer 
yeast  has  been  handed  down  from  brewery  to  brewery  from  time  im- 
memorial, and  that  other  industrial  yeasts  used  today  may  have  their 
beginning  in  early  Egyptian  history.  The  domestic  yeasts  by  con- 
tinued cultivation  by  man  may  have  been  reduced  to  a  constant  form 
of  a  yeast.  Such  is  not  the  case  with  wine  yeasts.  These  exist 
naturally  on  the  surface  of  the  grapes  and  it  is  only  necessary  to 
press  out  the  juice  which  will  soon  ferment.  But  where  does  this 
yeast  come  from  which  is  on  the  surface  of  the  grape? 

131 


132  ORIGIN   OF  THE  YEASTS 

Pasteur  l  was  one  of  the  first  to  attempt  to  answer  this  question. 
He  began  in  1875  a  series  of  investigations  to  find  out  whether  the 
yeasts  could  be  isolated  from  the  skin  of  the  grapes  and  whether 
they  were  present  only  at  one  time  of  the  year.  At  different  times 
in  the  year  he  placed  pieces  of  the  vine  and  grape  leaves  in  tubes  of 
sterile  wort.  This  experiment  indicated  that  during  the  autumn  the 
yeasts  existed  on  practically  all  parts  of  the  plant  and  that  they 
were  very  unequally  distributed  on  the  grapes  themselves.  He  fur- 
ther showed  that  the  yeasts  were  present  only  during  the  period  of 
maturity  in  the  grape,  and  that  it  was  not  present  at  other  times. 
The  yeasts  were  found  to  be  present  during  the  fall,  to  gradually 
disappear  during  the  winter. 

Where  do  these  yeasts  come  from?  In  what  form  do  they  pass 
the  winter?  The  problem  is  an  intricate  one.  Pasteur  has  remarked, 
however,  that  the  yeast  is  always  associated  with  another  fungus, 
Dematium  pullulans  which,  according  to  him,  is  present  on  the  grape 
vine  during  the  whole  year.  Pasteur  thus  thought  that  possibly  this 
Dematium  pullulans  developed  into  the  yeasts,  and  this  theory  seemed 
more  plausible  when  it  is  remembered  that  this  fungus  has  yeast-like 
stages  in  its  life  cycle.  This  idea  of  Pasteur's  corroborated  the 
assertions  of  the  botanist  Brefeld  for  whom  the  yeasts  were  only  de- 
velopmental forms  for  more  complex  fungi  as  the  Ustilagines.  Pas- 
teur expressed  it  thus:  "  The  yeast  cells  originate  from  little  brown 
bodies  (cysts  of  Dematium)  which  the  microscope  demonstrates  so 
abundantly  among  the  pollen  of  fruits."  Pasteur  soon  gave  up  this 
idea,  especially  when  the  celebrated  Chamberland  showed  that  these 
yeasts  of  Dematium  did  not  produce  alcoholic  fermentation. 

This  opinion  has  been  especially  maintained  by  Jorgensen.2  Ac- 
cording to  this  author  the  yeasts  spring  from  yeast-like  structures 
of  Dematium  pullulans  as  was  thought  by  Pasteur.  These  were  re- 
garded as  being  constantly  present  in  the  atmosphere,  and  on  the  parts 
of  grape  vines,  etc.  These  are  supposed  not  to  develope  into  the  true 
Saccharomyces  capable  of  producing  alcohol  and  forming  endospores. 

Somewhat  the  same  idea  is  expressed  by  Juhler  who  observed  a 
fermentation  in  a  flask  of  rice  starch  inoculated  with  Aspergillus 
oryzae  which  serves  the  Japanese  in  making  sake.  Jorgensen,  also, 
believed  a  relation  between  the  conidia  of  this  fungus  and  the  true 
yeast.  This  assertion  has  been  sustained  by  Sorel.3 

1  Pasteur.     Etude  sur  la  biere,  1876. 

2  Jorgensen,  A.     Der    Ursprung    der    Hefen.     Cent.    Bakt.    2,    1895.     Ueber 
Ursprung  der  Alkoholhefen.     Ber.  d.  Garungsphysiol.  Lab.  von  Jorgensen.      Copen- 
hagen, 1,  1895. 

3  Sorel.     Etude  sur  V Aspergillus  oryzae.     Comp.  Rend.  Acad.  Sciences,  121, 1895. 


HISTORICAL  133 

The  investigations  of  Seiter l  and  especially  those  of  Hansen, 
Klocker 2  and  Schionning  have  established  with  a  remarkable  pre- 
cision that  these  investigators  were  led  astray  by  impurities  in  their 
cultures.  They  have  shown  that  the  yeast-like  structures  of  Dema- 
tium  never  sporulate,  and  that  the  leavening  agent  of  sake  consists, 
besides  Aspergillus  oryzae,  of  a  yeast  in  no  way  related  to  the  mold 
which  induces  the  fermentation.  The  same  authors  have  made 
repeated  experiments  to  change  yeasts  into  molds  and  molds  into 
yeasts  under  conditions  such  as  those  which  obtain  in  nature.  They 
have  never  secured  reliable  results. 

However  the  question  of  the  origin  of  the  yeasts  from  molds  has 
been  raised  anew  by  the  investigations  of  Viala  and  Pacottet 3  on 
Gloeosporium  ampelophagum  and  Gloeosporium  nervisequum.  One  of 
these  fungi,  Gl.  nervisequum  presents  perithecia  which  have  been  ob- 
served by  Klebahn  who  has  classed  it  among  the  Ascomycetes  spheri- 
ceae.  The  other,  the  Gl.  ampelophagum,  on  account  of  the  presence  of 
pyknides,  organs  characteristic  for  the  Spheriaceae,  has  also  been 
placed  in  the  same  family  of  ascomycetes  although  perithecia  have 
never  been  observed.  According  to  Viala  and  Pacottet  these  two 
microorganisms  are  able  to  develop,  when  placed  in  suitable  nutrient 
media,  into  true  yeasts,  capable  of  setting  up  the  alcoholic  fermenta- 
tion and  forming  endospores  identical  in  all  respects  with  those 
formed  by  Saccharomycetes.  These  yeasts  become  fixed  after  culti- 
vation in  the  same  medium,  and  find  it  impossible  to  return  to  the 
mycelium  state.  Viala  and  Pacottet  conclude  that  yeasts  originate  at 
the  expense  of  more  highly  developed  fungi  and  think  that,  if  it  is  im- 
possible to  change  the  industrial  yeasts  into  mycelial  conditions,  it 
is  due  to  a  long  existence  in  the  state  of  yeasts  and  it  has  become  im- 
possible to  change.  Thus  according  to  these  investigators  the  yeast 
sporangium  may  not  possess  the  value  of  the  asc  but  the  endospores 
may  result  from  an  encystment  of  protoplasm  without  other  mor- 
phological significance. 

Guilliermond  4  in  studying  this  question  and  trying  to  reproduce 
the  results  of  Viala  and  Pacottet  established  that  impurities  in  cul- 

1  Seiter,  O.     Studien  ii.  d.  Abstammung  d.  Saccharomyceten.     Cent.  f.  Bakt. 
2,  1906. 

2  Klocker,  A.,  and  Schionning,   H.       Que  savons-nous  sur  1'origine  des  Sac- 
charomyceten.   C.  R.  du  Lab.  de  Carlsberg,  55,  1896. 

3  Viala,   P.  and  Pacottet,  P.     Nouvelles  recherches  sur  PAnthracnose.     Rev. 
de  viticulture,   1905.     Levures  et  kystes  des  Gloeosporium.      Ann.  Inst.  Agr.  5, 
1906. 

4  Guilliermond,   A.     A  propos  de  1'origine  des  levures.     Ann.  mycologici,   5, 
1907;    Recherches  sur  le  dev.  du  Gl.  nervisequum  et  sa  pretendue  transformation 
en  levures.     Rev.  gen.  de  Botanique,  20,  1908. 


134  ORIGIN  OF  THE  YEASTS 

tures  could  explain  the  results  of  these  two  workers.  In  repeating  the 
investigation  on  Gl.  nervisequum  he  showed  that  this  fungus  never 
produced  the  yeast-like  structures  when  grown  in  certain  media. 
On  the  other  hand,  he  established  that  the  yeasts  which  these  workers 
thought  they  observed  as  derivatives  of  GL  nervisequum  present  the 
same  characteristics  as  the  yeast-like  structures  of  a  species  of  De- 
mat  ium  which  he  observed  in  the  earlier  inoculations  of  the  Gloeo- 
sporium.  This  Dematium  exists  on  all  of  the  leaves  of  the  plane  tree 
and  develop,  almost  always,  in  a  state  of  impurity  in  the  first  arti- 
ficial cultures  of  GL  nervisequum.  Then,  to  such  data,  Guilliermbnd 
attributes  the  conclusions  of  Viala  and  Pacottet.  As  for  the  endo- 
spores  described  by  Viala  and  Pacottet  in  the  yeast  structures,  they 
may  have  been  simply  fat  droplets  from  old  cells  of  Dematium  which 
by  the  size  and  regular  positions  resemble  the  endospores  of  yeasts. 
Whatever  is  the  case,  it  is  definitely  established  that  GL  nervisequum 
does  not  form  yeasts 


(B)   Studies  in  Life  Cycles  of  Yeasts  in  Nature 

This  conclusion  on  the  transformation  of  yeasts  is  fully  confirmed 
by  the  careful  investigations  of  Hansen  l  on  the  life  cycles  of  yeasts. 
The  first  observations  of  this  author  date  back  to  1881,  and  are  con- 
cerned with  Saccharomyces  apiculatus.  This  yeast  is  particularly 
adapted  to  life  history  studies  on  account  of  the  special  form  of  its 
cells.  (Fig.  6.)  Hansen  observed  that  this  yeast  existed  on  many  dif- 
ferent fruits  and  that  it  was  found  only  on  the  walls.  It  was  only 
present  on  the  fruits  and  not  on  other  parts  of  the  plant.  It  appears, 
then,  that  it  lived  only  where  there  was  sugar  or  where  it  was  able  to 
multiply. 

Hansen  thought  that  the  rain  and  decay  of  the  plant  carried  this 
to  the  ground  on  which  fruit  trees  grow.  It  seems,  then,  that  this 
yeast  is  able  to  hibernate  in  soil  near  fruit  trees.  If  samples  of  this 
earth  are  taken  in  the  springtime,  S.  apiculatus  is  always  found.  Finally 
to  prove  this  hypothesis,  he  inoculated  soil  and  left  it  out  through 
the  winter.  From  time  to  time,  he  sampled  this  soil  and  always 
found  Saccharomyces  apiculatus.  Hansen  has  thus  demonstrated  that 
Saccharomyces  apiculatus  is  able  to  perpetuate  itself  in  the  soil  from 
year  to  year. 

1  Hansen,  E.  Recherches  sur  la  physiologic  et  la  morphologie  des  ferments 
alcooliques.  Sur  le  S.  apiculatus  et  sa  circulation  dans  la  nature.  Comp.  Rend, 
du  lab.  de  Carlsberg,  1,  Livr.  4.  1881.  Nouvelles  recherches  sur  la  circulation  du 
S  apiculatus  dans  la  nature.  Ann.  des.  Sc.  nat.  et  botanique,  7th  Series,  1890. 


STUDIES   OF  LIFE   CYCLES   IN   NATURE  135 

On  the  other  hand,  he  has  shown  that  it  passes  the  winter  in  the 
soil,  for  he  examined  other  substances  such  as 'dust,  dried  fruit,  ani- 
mal excrement  and  never  found  this  yeast. 

The  investigations  of  Muller-Thurgau l  and  Berlese2  indicate 
somewhat  the  same  things.  Berlese  has  found,  in  April  and  June, 
Saccharomyces  apiculatus,  ellipsoideus  and  Pastorianus  in  the  earth 
of  vineyards  and  orchards.  These  yeasts  were  found  down  to  12  or 
13  centimeters  in  depth  and  seemed  to  be  equally  distributed  in  both 
sunny  and  shady  places.  This  is  interesting  for  it  shows  the  resist- 
ance of  these  yeasts  to  sun  and  light.  Berlese  has  also  found  S. 
apiculatus  on  the  bark  of  oak  and  olive  trees,  and  also  in  the  nectar 
of  flowers. 

Hansen3  has  undertaken,  in  recent  years,  a  study  of  the  life  cycle 
of  yeasts  in  nature  to  find  out  whether  all  of  the  yeasts  behave  like 
S.  apiculatus.  He  used  various  yeasts  in  this  investigation  and 
experienced  some  difficulty,  for  the  shapes  of  the  various  yeasts  did 
not  lend  themselves  to  a  ready  recognition.  They  were  very  easy 
to  confuse  with  the  yeast  forms  of  Dematium  and  other  fungi.  Only 
one  character  was  available  and  that  was  the  formation  of  endospores. 

In  his  recent  investigations,  Hansen  investigated  the  presence 
of  yeasts  in  the  soil  about  Copenhagen  and  whether  they  were  present 
at  all  periods  of  the  year.  These  environs  included  many  orchards, 
gardens  and  vineyards.  He  was  scarcely  able  to  find  a  spot  which 
did  not  contain  yeast.  They  were  almost  always  present  in  the  sur- 
face layers  and  scarcely  at  all  in  the  deeper  layers;  at  all  times  of 
the  year  he  was  able  to  isolate  them.  The  soil  in  vineyards  and  or- 
chards was  plentifully  supplied  with  them  but  they  diminished  in 
numbers  as  one  went  away  from  the  orchards.  Thus,  in  100  analyses 
of  soil  under  fruit  trees,  67  showed  the  presence  of  yeasts;  away  from 
such  places  in  fields,  only  19  per  cent  of  the  samples  indicated  the 
presence  of  yeasts. 

Hansen  has  also  observed  yeasts  in  the  soils  of  beech,  fir,  pine 
and  oak  groves  but  much  less  numerous  than  in  fruit  groves.  Only 
30  per  cent  of  the  samples  yielded  the  presence  of  yeasts.  Such 
yeasts  belonged  to  special  genera  such  as  Pichia  membranefaciens 
and  Willia  anomala. 

1  Muller-Thurgau,  H.     Ueber  den  Ursprung  der  Weinhefe.     Weinbau  Weinh. 
No.  40  and  41,  1889. 

2  Berlese,  A.     Verhalten  des  Saccharomyceten  an  den  Weinstocken  iiber  die 
Verteilung  der  alkoholischen  Fermenten  in  der  Nature.     Ueber  die  Transport- 
mittel  der  alkoholischen  Fermente.    Rivista  di  patol.  vegetale  5,  1897. 

3  Hansen,  E.  C.     Neue  Untersuchungen  iiber  den  Kreislauf  der  Hefenarten 
in  der  Natur.     Cent.  Bakt.  10,  1903.     Ueber  die  Brutstatten  der  Alkolholgarungs- 
pilze  oberhalf  der  Erde.    Cent.  Bakt.  14,  1905. 


136  ORIGIN   OF  THE   YEASTS 

These  investigations  indicate  that  all  of  the  yeasts  studied  by 
Hansen  have  a  life  history  identical  with  that  of  S.  apiculatus.  The 
yeasts  hibernate  in  the  soil.  They  seem  to  differ  only  in  their  distri- 
bution. Hansen  explained  this  on  the  basis  of  spore  formation,  as- 
suming that  yeast  which  formed  no  spores  would  be  killed.  On  the 
other  hand,  thanks  to  the  presence  of  spores,  the  yeasts  live  a  longer 
time  than  S.  apiculatus  in  the  ground  water  which  carries  them  for 
longer  or  shorter  distances. 

It  is  then  necessary  to  determine  the  method  by  which  the  yeasts 
are  transferred  from  the  soil  to  the  fruit  skins.  Transportation 
through  the  air  seems  to  play  an  important  role.  Chamberland  has 
observed  that  there  are  many  yeasts  in  the  air  especially  during  sum- 
mer and  autumn.  One  may  detect  them  at  the  other  seasons  but 
they  are  not  so  common.  From  this  the  yeasts  seem  to  be  less 
abundant  during  the  rainy  seasons  of  the  year.  Hansen  l  states  that 
yeasts  are  always  found  in  the  atmosphere  but  in  different  numbers. 
Their  number  seems  to  be  increased  during  June  to  August  and  es- 
pecially at  the  beginning  of  September.  During  the  other  seasons, 
one  may  not  find  them  as  readily.  Berlese  did  not  find  any 
yeasts  in  the  air  during  April  and  May  but  was  able  to  find  S. 
apiculatus  in  the  beginning  of  June  and  during  July.  Thus  it  seems 
that  the  air  may  be  an  important  factor  in  transporting  the  yeasts 
from  the  ground  to  the  fruit.  On  this,  they  find  a  higher  tempera- 
ture and  more  favorable  environment  and  develop  to  maturity.  The 
presence  of  yeasts,  then,  in  the  air  seems  to  be  a  function  of  two  fac- 
tors: first,  an  active  development  of  these  organisms  on  the  skin  of 
the  fruit  and,  secondly,  an  absence  of  rain. 

Boutroux  2  has  shown  that  insects  play  an  important  role  in  the 
distribution  of  yeasts.  He  disclosed  the  presence  of  yeasts  on  various 
insects  (mosquitoes,  wasps,  bees,  gnats  and  ants).  Saccharomyces 
cerevisiae,  ellipsoideus  and  Pastorianus  were  demonstrated.  Wort- 
mann  and  Berlese  have  observed  the  same  things  and  regard  the  insects 
as  the  important  mode  of  distribution  of  yeasts  from  grape  to  grape 
and  from  vine  to  vine.  In  this  way,  Berlese  explains  the  presence  of 
S.  apiculatus  in  the  nectar  of  flowers  which  has  been  visited  by 
Vespa  crabro  in  which  he  has  observed  the  same  yeast.  He  does  not 
regard  the  deposition  of  the  yeasts  by  the  insects'  feet  with  much  favor 
but  points  out  that  the  yeasts  are  able  to  pass  through  the  intesti- 

1  Hansen,  E.  C.     Recherches  sur  les  organismes  qui  a  differentes  epoques  de 
1'annee,  se  trouvent  dans  1'air  a  Carlsberg  et  aux  alentoirs.     Comp.  Rend,  du 
lab.  de  Carlsberg,  1,  1882. 

2  Boutroux,  L.     Sur  1' habitat  et  la  conservation  des  levures  apontanees.     Bull, 
de  la  Soc.  Linn,  de  Normandie,  3rd  Series,  7,  1883;  Ann.  des  Sc.  nat.  Bot.  17,  1884. 


MORPHOLOGICAL,  CYTOLOGICAL   INVESTIGATIONS     137 

nal  canal  without  harm.  The  intestinal  canals  of  certain  diptera 
seem  to  be  the  normal  habitat  for  certain  yeasts;  in  fact,  he  has  ob- 
served S.  apiculatus  and  ellipsoideus.  Such  conclusions  are  in  accord 
with  the  work  of  Neumayer,  who  has  demonstrated  that  yeasts  are 
very  resistant  to  digestive  juices.  It  is  well  to  point  out  that  this 
means  of  dissemination  is  not  mentioned  by  other  authors,  Hansen, 
for  instance. 

(C)    Morphological  and  Cytological  Investigations  on  Yeasts 

It  has  just  been  stated  that,  under  no  circumstances,  are  we  able 
to  transform  yeasts  into  molds,  or  a  mold  into  a  true  yeast;  this  has 
not  been  observed  in  nature.  Hansen  did  not  hold  this  view  and  re- 
garded the  yeasts  as  an  autonomous  group  of  fungi,  Ascomycetes. 

Such  an  hypothesis  was  not  a  new  one.  Before  this,  Reess  and 
de  Bary  had  suggested  this  idea  and  noticed  the  superficial  similarity 
between  the  asc  of  the  yeasts  and  the  sporangium  of  the  molds.  The 
asc  is  a  single  character  which  distinguishes  between  the  true  yeasts  and 
yeast-like  structures  of  other  fungi.  So  little  was  known,  then,  about 
the  cytological  characteristics  of  the  asc  that  it  was  difficult  to  make 
any  definite  statements. 

For  a  long  time  this  morphological  problem  remained  untouched. 
Were  the  sporangia  of  yeasts  similar  to  the  ascs  of  the  Ascomycetes 
as  was  maintained  by  Hansen?  Or,  should  we  regard  them  as  approach- 
ing more  closely  the  sporangia  of  the  Mucors,  as  was  thought  by 
Brefeld?  Do  the  yeasts  represent  a  bona-fide  group  of  fungi  or  are 
they  developmental  forms  of  the  molds?  These  questions  remained 
unanswered.  One  may  always  suppose  that  the  yeasts  resulted  from  the 
molds  by  some  process,  hitherto  unobserved,  and  that  they  have  lost 
the  possibility  of  returning  to  the  state  of  a  mycelium.  We  have 
negative  proofs  in  favor  of  the  autonomy  of  the  yeasts. 

But  in  these  later  times,  new  facts  have  been  discovered.  It  has 
been  shown  in  the  preceding  chapter  that  the  cytological  studies 
on  the  asc  and  the  discovery  of  sexuality  in  yeasts  have  furnished 
definite  proof  of  the  ascogenous  nature  of  the  sporangia  of  yeast,  and 
have  proven  the  relationship  of  the  fungi  to  the  Ascomycetes.1 

1  It  might  be  well  to  point  out  that  what  distinguishes  the  group  of  Ascomy- 
cetes is  their  possession  of  an  aso  enclosing  from  4  to  8  ascospores.  The  asco- 
spores  are  differentiated  on  the  interior  of  the  asc  only  at  the  expense  of  part  of 
the  protoplasm.  The  rest,  or  epiplasm,  is  absorbed  by  the  ascospores  when  they 
develop.  Among  the  lower  ascomycetes  (Endomycetes)  the  ascs  form  only  at 
the  expense  of  terminal  cells  on  the  filaments.  Among  the  higher  Ascomycetes, 
they  are  united  in  great  numbers  in  organs  called  perethecia. 

A  sexual  process,  rudimentary  in  certain  types,  always  intervenes  in  the  origin 
of  the  yeasts.  Among  the  Exoascee  there  is  a  simple  nuclear  fusion.  Among  the 


138  ORIGIN   OF  THE  YEASTS 

The  investigations  of  Guilliermond  l  have  indicated  that  by  the 
morphological  and  cytological  characteristics,  the  sporangium  of  the 
yeasts  presents  a  remarkable  similarity  to  the  ascs  of  the  Ascomycetes. 
The  ascospores  develop  by  the  same  process. 

The  ascospores  in  certain  yeasts  present,  on  the  other  hand, 
characteristic  forms  absolutely  analogous  to  the  ascospores  of  cer- 
tain Ascomycetes.  Thus  it  is  that  the  ascospores  of  Willia  anomala 
are  identical  with  those  of  Endomyces  decipiens,  Endomyces  fibul'ger 
and  Ascoidea  rubescens.  Those  of  Willia  saturnus,  Schwanniomyces 
occidentalism  Debaromyces  globosus,  Monospora  cuspidata,  Nematospora 
coryli  have  forms  which  suggest  very  strongly  those  of  certain  ascomy- 
cetes.  Without  doubt,  the  number  of  ascospores  in  the  sporangium 
of  a  yeast  is  variable  although  it  is  constant  for  an  asc.  How- 
ever, one  notices  that  the  number  of  ascospores  tends  to  become 
fixed  in  an  asc  in  most  of  the  yeasts  while  with  some,  it  remains 
variable.  Thus  it  is  that  in  Schizosaccharomyces  the  number  4  or  8 
is  usually  seen.  In  Saccharomyces  Ludwigii  the  ascospores  are  con- 
stantly present  to  the  number  of  4.  Even  in  those  cases  in  which 
this  varies,  there  is  a  slight  tendency  for  it  to  become  fixed. 

Finally,  the  discovery  of  a  copulation  in  the  origin  of  the  asc  in 
Schizosaccharomyces,  the  Lygosaccharomyces  and  Debaromyces  globo- 
sus which  absolutely  resembles  that  of  certain  Ascomycetes  (Bremas- 
cus  and  End.  Magnusii)  furnishes  a  strong  argument  in  favor  of 
their  homologation.  The  existence  of  this  copulation,  together  with 
morphological  and  cytological  characteristics  of  ascs  of  yeasts,  suffices 
to  demonstrate  their  place  with  Ascomycetes.  The  question  of  the 
origin  and  systematic  relationship  of  the  yeasts  is  definitely  settled 
today.  The  Saccharomycetes  constitute  an  autonomous  group  of  lower 
Ascomycetes.  It  has  been  stated  that  among  the  true  yeasts  which 
form  ascs,  there  are  some  which  do  not  sporulate;  such  are  the  My- 
coderma  and  Torula.  But,  as  will  be  pointed  out  further  on,  many 
of  the  yeasts  are  able  on  account  of  special  conditions,  to  definitely 
lose  their  property  of  sporulating.  It  is  possible  that  these  are  true 
Saccharomycetes  having  become  asporogenous  but  it  is  also  possible 
that  they  are  derived  forms  from  molds  fixed  in  the  state  of  yeasts. 
The  question  of  their  origin  and  their  position  in  classificatory  sys- 
tems is  then  not  settled.  Ought  we  to  separate  the  family  of  Sac- 
charomyces in  which  are  the  true  yeasts? 

higher  Ascomycetes,  less  is  known.  According  to  Harper,  it  consists  of  a  true 
copulation  to  give  the  perethecia;  according  to  Dangeard,  it  is  simply  a  nuclear 
fusion.  The  question  is  still  very  obscure.  Among  the  Endomyces,  copulation 
is  very  clear. 

1  Guilliermond,  A.     L'origine  des  levures.     Annales  mycologici,  5,  1907. 


PHYLOGENY   OF  'THE  YEASTS  139 

Phylogeny  of  the  Yeasts.  Their  Affinities  in  the 
Group  of  Ascomycetes 

What  place,  in  the  classification  of  the  Ascomycetes,  shall  the 
yeasts  occupy,  what  are  their  relationships  to  the  other  Ascomycetes? 
We  shall  now  take  up  that  question.1  Up  to  recent  times,  it  seemed 
incapable  of  being  answered. 

The  species  of  Exoascus  are  filamentous  fungi,  in  which  certain 
terminal  cells  form  octosporous  ascs  after  a  fashion  comparable  to 
those  formed  by  the  yeasts.  The  ascospores  germinate,  producing 
generations  of  yeasts.  It  is  evident  that,  by  the  characters  of  their 
ascs  and  the  shape  of  the  yeast-like  structures  to  which  they  give 
birth,  they  are  like  the  Saccharomyces.  They  differ  in  the  presence 
of  a  typical  mycelium.  But  we  have  seen  that  the  yeasts  themselves, 
under  certain  conditions  are  able  to  manifest  a  tendency,  more  or 
less  marked,  to  vegetate  with  a  mycelium.  The  investigations  of 
Dangeard  and  Ikeno  have  shown  that  the  asc  in  Exoascus  possesses 
two  nuclei  at  the  time  of  its  formation,  and  these  fuse  into  one  hav- 
ing the  nuclear  divisions  necessitated  by  8  ascospores.  Dangeard 
regards  this  fusion  as  karyogamy  and  the  equivalent  of  fecundation 
but  the  interpretation  of  this  process  remains  very  much  discussed. 
The  yeasts  are  closely  distinguished  from  Exoascus  in  that  they  show 
no  nuclear  fusion  in  the  asc.  It  is  true  that  in  a  few  varieties,  the 
asc  results  from  a  true  copulation  but  in  all  of  the  varieties  in  which 
this  phenomenon  is  lacking  one  is  unable  to  detect  nuclear  fusion. 
Then,  from  this  point  of  view,  the  yeasts  resemble  Exoascus. 

On  the  other  hand  it  has  been  noticed  for  a  long  time  that  the 
family  of  Endomyces  includes  varieties  related  to  the  yeasts.  But 
our  information  with  regard  to  this  group  has  remained  very  vague. 

Recent  investigations  by  Guilliermond l  have  allowed  us  to  fill 
this  gap  in  our  knowledge  and  -at  the  same  time  determine  the  sys- 
tematic relationships  of  the  yeasts.  The  results  of  these  investiga- 
tions are  sufficiently  important  to  receive  more  extended  treatment 
at  this  time.  The  family  of  Endomycetes  presents  only  a  small  num- 
ber of  representatives  in  which  the  genera  Eremascus  and  Endomyces 
are  best  known.  We  shall  take  up  some  of  the  shapes  of  these  genera. 

Only  two  genera  of  Eremascus,  the  E.  albus  and  E.  fertilis,  re- 
cently discovered  by  Stoppel,  have  been  known  up  until  recently. 
The  former  is  not  well  known;  the  latter  has  been  subjected  to  a 
conscientious  investigation  by  Stoppel  whose  results  were  confirmed 
by  Guilliermond.  The  mycelium  of  E.  fertilis  presents  cells  which 

1  Guilliermond,  A.  Recherches  cytologiques  et  taxonomiques  sur  les  En- 
domycet<§es.  Rev.  g.  de  Botan.  26,  1909. 


140 


ORIGIN   OF  THE  YEASTS 


are  generally  mononuclear.  It  never  produces  conidia  but,  on  the  con- 
trary, forms  a  rather  large  number  of  ascs.  These  are  derived  from 
an  isogamic  copulation  which  is  accomplished,  usually,  between  two 
contiguous  cells  in  the  same  filament.  The  two  cells  unite  by  means 
of  little  canals  playing  the  role  of  gametes,  which  anastomose,  form- 
ing in  this  way,  a  sort  of  bridge  between  the  two  cells.  (Fig.  54.) 
The  wall  which  separates  the  two  cells  at  the  middle  of  the  copula- 
tion canal  is  not  slow  to  break  down.  Part  of  the  cytoplasm  enters 

the  canal  from  each  cell  and  forms 
a  swelling  at  the  middle  of  the 
copulation  canal  which  becomes 
the  zygospore.  At  this  moment  each 
of  the  cells  divides  its  nucleus. 
One  of  the  daughter  nuclei  thus 
formed  remains  in  the  cell  and  the 
other  passes  into  the  zygospore. 
(Fig.  54.)  There  the  two  sexual 
nuclei  fuse  and  develop  into  a  single 
large  one.  As  this  proceeds  the 
zygospore  forms  a  wall  which  sepa- 
rates it  from  the  two  threads  which 
formed  it.  From  this  the  zygo- 
spore grows  and  develops  into  an 
octosporous  asc  quite  similar  to  that 
of  a  yeast.  The  ascospores  are 

in  'Eremascus  fertilis;  1  and  2:J3e-     enveloped  as  those  of  Saccharomyces 

guttulatus  by  a  double  membrane 
in  which  the  external  one  breaks 
at  the  moment  of  germination. 
They  germinate  directly  into  a 
mycelium.  It  cannot  be  refuted  that  Eremascus  resembles  the  yeasts; 
its  ascs  present  the  same  characteristics  as  those  of  the  yeasts  and 
result  from  a  copulation  which  is  able  to  be  approached  by  cells 
which  one  sees  in  the  Zygosaccharomyces  and  Schizosaccharomyces. 
By  the  copulation  which  precedes  the  formation  of  the  ascs,  many 
yeasts  are  similar.  In  most  of  the  yeasts,  it  is  true,  copulation  differs 
from  that  of  E.  fertilis  in  that  it  is  incomplete  and  ends  in  the  for- 
mation of  an  asc  having  the  form  of  a  dumb-bell ;  Schizosaccharomyces 
octosporus  offers  an  intermediate  stage  between  the  copulation  of  Ere- 
mascus and  that  of  the  yeasts.  In  this  yeast,  copulation  is  more 
often  complete  and  produces  a  large  oval  cell  which  is  transformed 
into  an  asc.  In  this  case,  copulation  is  absolutely  homologous  to 
that  of  E.  fertilis.  In  reality,  E.  fertilis  differs  especially  from  the 


Fig.    54.  — Different    Stages    in    the 
Copulation  and  Formation  of  Ascs 


tion;  5:  Demarcation  of  the  Asc; 
6  and  7:  Formation  of  the  Asco- 
spores. 


PHYLOGENY  OF  THE  YEASTS 


141 


55.  —  Mycelium 
in  Endomyces  fibu- 
liger  Forming  a 
Number  of  Yeast- 
Like  Bodies. 


yeasts  in  that  yeasts  are  reduced  to  the  state  of  isolated  cells  while 
the  E.  fertilis  remains  in  a  mycelial  condition. 

With  the  genus  Endomyces,  one  begins  to  approach  the  yeasts. 
Endomyces  fibuliger,  discovered  by  Lindner,  shows  striking  resem- 
blances to  Eremascus  fertilis.  It  differs,  however, 
by  that  fact  that  the  mycelium  formed  from 
uninuclear  cells  gives  birth,  by  budding,  to  a 
series  of  yeast-like  structures  (Fig.  55)  which  sug- 
gests that  this  fungus  is  intermediary  between 
the  yeasts  and  Eremascus.  Under  certain  con- 
ditions, it  is  able  to  vegetate  exclusively  with  the 
form  of  yeasts.  E.  fibuliger,  on  the  other  hand, 
produces  conidia  which  form  themselves  by  bud- 
ding and  are  able  to  be  compared,  to  a  certain  Fig 
extent,  with  the  "durable  cells"  of  yeast.  Finally, 
it  furnished  numerous  ascs  very  similar  to  those 
of  Eremascus  which  contain  only  4  ascospores. 
These  ascs  are  formed  often  simply  by  budding  of  the  elements,  but 
in  many  asces,  they  form  after  attempts  at  copulation  at  the  expense 

of  an  anastomosis  which  occurs  be- 
tween two  neighboring  cells  taking 
place  in  the  following  manner:  Two 
units  of  the  mycelium  send  out  little 
rootlets.  These  anastomose  but  the 
wall  which  is  formed  between  them 
does  not  break  down  and,  in  many 
cases,  there  is  no  mixture  of  the  cell 
contents.  Generally  one  of  the  pro- 
tuberances stops  developing,  the  other 
elongates,  bends  itself  toward  the  first 
and  forms  by  a  swelling,  a  tetrasporous 
asc.  (Figs.  56,  1,  2,  3,  5,  and  6.)  In 
the  meantime  the  two  rootlets  develop 
into  an  asc.  In  some  cases,  the  two 
protuberances  progress  side  by  side, 
Fig.  56. —Different  Stages  in  the  without  anastomosis,  each  forming  a 

swelling  which  becomes  the  mother 
cell  of  an  asc;  these  two  cells,  thus 
formed,  bind  themselves  one  to  the  other  by  a  sort  of  copulation 
canal  in  which  the  wall  is  not  broken  down.  It  also  happens  that 
the  extremities  of  a  filament,  formed  by  the  walling  off  of  a  chain  of 
cells  which  swell  up,  transforms  itself  into  an  asc.  Often,  in  this 
case,  anastomosis  is  often  noticed  binding  the  ascs  two  by  two. 


142 


ORIGIN  OF  THE  YEASTS 


Fig.  57.  —  A.  Showing  Copulation  and  Asc  Formation 
in  Eremascus  fertilis;    B, 


fibuliger. 


These  anastomoses  prove  then,  that,  although  sexuality  may  have 

disappeared,  there  seems  to  be  a  rudimentary  sexual  attraction  quite 

comparable  to  the  phenomena  which  have  been  observed  in  certain 

yeasts  (Schw.  occidenta- 
lis,  yeasts  of  Rose  and 
Dombrowski,  etc.). 
However  when  one 
compares  these  anas- 
tomoses with  the  sexual 
production  of  Eremas- 
cus fertilis,  he  is  struck 
by  the  resemblance 
which  exists  between 
the  method  of  forma- 
tion of  ascs  in  these 
two  fungi  (Fig.  57). 

The  Same  for  Endomyces  In  one  and  the  other, 
two  contiguous  cells 

produce  protuberances  which  seem  to  search  for  each  other.     With 

Eremascus  fertilis,  they  reunite 

to   form   an  egg  while    in    E. 

fibuliger  they  constantly  fail  in 

their    attempt.      (Fig.    57,    A 

and  B.)    It  is  not  doubtful  that 

the  anastomosis  which  precedes 

the  formation  of  the  asc  in  the 

latter  fungus  represents  traces 

of    an    ancestral    reproduction 

analogous  to  that  which  occurs 

in  Eremascus  fertilis  to  which 

E.  fibuliger  is  closely  related. 

We  may  then  regard  E.  fibuliger 

as  a  form  derived  from  a  genus 

neighboring  Eremascus  fertilis. 

The  ascospores  have  the  same 

form  as  those  of  Willia  anomala; 

they  are  hemispherical  and  pro- 
vided with  a  projecting  color 

giving  them  the  appearance  of 

a  hat.      On    the    other    hand, 

they  are  supplied,  like  those  of  E.  fertilis,  with  two  membranes.     The 

external  membrane  is  burst  during  germination.  The  ascospores  ger- 
minate either  in  the  form  of  yeasts  or  with  a  mycelium. 


Fig.  58.  —  Endomyces  capsularis. 

1,  Fragment  of  the  Mycelium  Showing  the  Formation 
of  Yeasts.  2  and  3,  Fragment  of  the  mycelium  Pro- 
ducing Ascs. 


PHYLOGENY  OF  THE  YEASTS 


143 


Endomyces  fibuliger  l  constitutes  a  link  between  Eremascus  fertilis 
and  Endomyces  Hordei  recently  described  by  Saito.  This  last-men- 
tioned species  has  the  same  characteristics  as  Endomyces  fibuliger 
with  the  exception  that  no  conidia,  but  only  yeast  forms,  are  found. 
It  forms  ascospores  in  the  shape  of  a  hat  but  these  ascs  result  from 
simple  budding  of  the  mycelium  without  presenting  an  anastomosis. 
All  traces  of  sexuality  have  disappeared.  The  ascospores  have  a 
double  membrane  and  germinate  by  simple  budding.  Endomyces 
Hordei  represents  a  higher  step  in  the  parthenogenetic  evolution  than 
seems  to  have  taken  place  in  the  descendants  of  Eremascus. 

With  Endomyces  capsularis,  discovered 
a  few  years  ago  by  Schionning2  we  have 
a  similar  species  but  one  more  closely  con- 
nected to  the  yeasts.  This  fungus  also 
has  a  branching  mycelium  with  septa 
made  of  cells  with  one  nucleus  and  which 
form  numerous  yeast  bodies  by  budding. 
These,  however,  are  much  larger  in  number 
than  in  Endomyces  fibuliger  and  Endomyces 
Hordei.  Endomyces  capsularis  also  has 
ascs  with  its  ascospores  possessing  a 
double  membrane  and  germinating  either 
into  yeast  bodies  or  a  mycelium.  The 
ascs  are  formed  as  in  Endomyces  Hordei 
by  a  sort  of  budding  of  the  cells  or  of  any 
cell  in  the  mycelium  without  any  anas- 
tomosis. 

End.  javanensis,  described  by  Klocker,3 
offers  a  form  of  transition  more  disputed 
between  Endomyces  and  the  yeasts.  The 
mycelium  is  greatly  reduced  and  yeast  forms  predominate.  The  ascs, 
always  parthenogenetic,  form  indifferently  at  the  expense  of  some 
cells  in  the  mycelium  or  of  a  yeast  cell.  They  include  a  single  asco- 
spore  much  like  the  ascospore  of  Sch.  occidentalis.  They  germinate 
either  into  the  form  of  yeasts  or  a  mycelium.  This  fungus  presents, 
then,  such  great  resemblances  to  the  yeasts  that  it  is  difficult  to  know 
whether  it  should  be  classed  among  the  Endomyces  or  among  the 

1  Lindner,  P.    Endomyces  fibuliger,  n.  sp.  einer  neuer  Garungspilze  und  Erzeuger 
des  sog.  Kreidekrankheit  des  Brotes.    Woch.  Baruerei,  24,  1908. 

2  Schionning.    Nouveau   genre   de   la  famille   des  Saccharomycetees.     Comp. 
Rend.  trav.  lab.  Carlsberg,  6,  1908. 

3  Klocker,  A.     Endomyces  javanensis,  nov.  sp.     Comp.  Rend,  des  trav.  du 
lab.  de  Carlsberg,  6,  1909. 


g.  59.  —  Fragment  of  Myce- 
lium in  Endomyces  Magnusii 
in  the  Process  of  Breaking 
into  Oidia  (after  Rose). 


144  ORIGIN   OF  THE   YEASTS 

yeasts.  Nevertheless  since  the  essential  characteristic  of  the  genus 
Endomyces  is  the  presence  of  a  typical  mycelium  from  which  the  ascs 
spring  exclusively,  it  seems  that  E.  javanensis  ought  to  be  regarded  as 
a  yeast. 

The  information  with  regard  to  these  various  fungi  explains  anew 
the  phylogeny  of  the  yeasts.  Indeed,  it  is  possible  to  regard  the 
genus  Eremascus  as  an  ancestral  form.  From  this  may  originate  a 
form,  quite  hypothetical,  related  to  Endomyces  fibuliger,  but  differ- 
ing by  the  existence  of  an  isogamic  copulation  characteristic  of  Ere- 
mascus. This  copulation  which  is  reduced  to  an 
unfruitful  attempt  with  E.  fibuliger  has  completely 
disappeared  in  E.  capsularis.  From  this  hypothetical 
form  the  yeasts  may  derive  by  regression  and  from 
the  mycelial  form  which  yields  its  place  to  yeast-like 
forms. 

Summarizing,   this  hypothetical   fungus,  derived 
Endomyces   deci-  from    Eremascus,    may   be    the   beginning  of    two 
piens    (after    de  branches,  one  with  E.  fibuliger  and  the  E.  capsularis, 
the  other  with  Zygosaccharomyces  and  the  Saccharo- 
myces.     The  genus  Saccharomyces  represents  a  parthenogenetic  form 
derived  from  Zygosaccharomyces. 

Now  it  remains  to  determine  the  origin  of  the  Schizosaccharo- 
myces.  The  study  of  two  other  forms  of  the  genus  Endomyces,  the 
E.  Magnusii  and  the  E.  decipiens,  has  given  some  information  on  the 
subject. 

These  two  fungi  resemble  very  much,  in  the  whole  of  their  develop- 
ment, E.  fibuliger;  but  they  are  closely  distinguished  by  the  fact 
that,  in  place  of  producing  yeast-like  bodies,  they  form,  by  dissocia- 
tion of  their  mycelium,  cells  called  oidia  which  are  capable  of  dividing 
transversely  like  the  cells  of  Schizosaccharomyces.  (Fig  59.)  Let  us 
state  that,  in  certain  media,  a  true  mycelium  is  not  formed  but 
almost  always  oidia  which  multiply  like  the  Saccharomyces.  In  its 
general  form,  the  oidium  is  identical  to  the  cell  of  Schizosaccha- 
romyces. Cytologically,  however,  it  differs  more  often  in  E.  Magnusii 
by  the  presence  of  many  nuclei.  Nevertheless,  many  of  the  oidia 
of  E.  Magnusii  offer  only  a  single  nucleus  and  Dangeard  has  shown 
that  in  the  oidia  of  E.  decipiens,  this  is  always  the  case. 

These  two  fungi  present  also  chlamodyspores  which  are  formed  like 
oidia  by  a  sort  of  dissociation  of  cells  in  the  mycelium  but  are  dis- 
tinguished by  the  formation  of  a  very  thick  membrane  and  by  the 
fact  they  cease  to  divide  until  they  find  conditions  sufficiently  favor- 
able. These,  then,  are  sort  of  encysted  oidia  and  may  be  compared 
to  the  durable  cells  of  yeasts. 


PHYLOGENY  OF  THE  YEASTS 


145 


Finally,  E.  decipiens  and  E.  Magnusii  produce  numerous  ascs 
which  form  at  the  extremities  of  the  filaments.  In  E.  decipiens,  they 
are  not  preceded  by  a  sexual  act,  but  in  E.  magnusii  a  heterogamic 
copulation  has  been  established  which  results  in  the  asc.  (Fig.  6.) 
This  is  accomplished  between  a  male  gamete  and  a  female  gamete, 
each  being  at  the  end  of  a  filament.  The  male  gamete  is  a  small, 
short,  cell,  with  a  single  nucleus  which  is  located  at  the  end  with  a 
shape  like  a  screw.  The  female  gamete  is  a  long  cell  which  also  in- 


Fig.  61.  —  Various  Stages  in  the  Copulation  and  Formation 
of  Ascs  in  Endomyces  Magnusii. 

eludes  a  single  nucleus.  The  gametes  unite  by  their -ends.  (Fig.  61, 
1  and  2.)  The  middle  wall  which  separates  them  breaks  down, 
their  contents  fuse  protoplasm  with  protoplasm,  nucleus  with  nucleus. 
The  egg  thus  formed  grows  and  is  transformed  into  a  tetrasporous 
asc.  Although,  heterogamic,  this  copulation  resembles  very  much 
that  of  Sch.  octosporous. 

It  looks  as  if  one  might  regard  the  Schizosaccharomyces  as  de- 
rived from  a  hypothetical  form  analogous  to  E.  Magnusii  but  more 
advanced,  in  which  the  copulation  may  be  isogamic.  From  this  form 
comes  on  one  side  E.  Magnusii  and  its  parthenogenetic  form,  E. 
decipiens,  and  on  the  other  part  Schizosaccharomyces. 

The  scheme  presented  below  represents  the  different  steps  in  the 
phylogeny  of*  the  yeasts  according  to  the  theory  which  has  been  out- 


146  ORIGIN  OF  THE  YEASTS 

lined.  The  budding  yeasts  are  sprung  from  a  hypothetical  form, 
Endomyces  a,  analogous  to  End.  fibuliger  but  have  kept  the  copula- 
tion of  Eremascus.  This  copulation  persists  in  the  Zygosaccharomyces, 
no  trace  of  it  remains  in  the  Schwanniomyces,  disappears  completely 
in  the  Saccharomyces  and  is  replaced  by  a  parthenogamy  between  the 
ascospores  in  the  yeast  Johannisberg  II.  The  Schizosaccharomyces 
spring  from  a  hypothetical  form,  Endomyces  6,  related  to  End.  Mag- 
nussii  but  with  isogamic  copulation.  The  Schizosaccharomyces  seem, 
like  other  yeasts,  to  be  more  advanced  toward  parthenogenesis  as  is 
evidenced  by  a  variety,  Sch.  mellacei,  which  has  lost  its  sexuality. 

.Eremascus  fertilis 

Endomyces  b _/  >.   • 

^f  VEndomyces  a 

Endomyces  MagnusH, 


Endomyces  •"oecipiens 


Schizosaccharomyces/  octosporus 


Schizosaccharomyces/  Pombe 


Schizosaccharomyces 
mellacei 


Indomyces  fibuliger 

Endomyces  capsularis 
Zygosaccharomyces 

Schwanniomyces 
Saccharomyces 


Parthenogenetic  variety  \ 

•  Yeast  Johannisberg  H 

Summarizing,  it  seems  proper  to  consider  the  Saccharomyces  and 
other  budding  yeasts  and  the  Schizosaccharomyces  as  derived  from  a 
form  related  to  Eremascus  fertilis.  From  this  common  stock,  two 
branches  spring:  one  which  forms  the  E.  Magnusii  and  Schizosaccha- 
romyces, the  other  which  forms  E.  fibuliger,  the  Zygosaccharomyces 
and  the  budding  yeasts.  The  question  of  the  phylogeny  of  the  yeasts 
may  be  considered  today  as  a  little  more  settled.1 

1  Another  theory  has  been  recently  proposed  by  Nadson  following  his  dis- 
covery of  Nadsonia  fulvescens.  According  to  this  author  the  Endomycelaceae 
and  the  Saccharomycetaceae  represent  degraded  forms  derived  from  the  higher 
Ascomycetes.  The  yeasts  possess  a  copulation  in  the  germination  of  the  ascospores 
with  Saccharomyces  Ludwigii  being  an  archaic  yeast.  This  theory  lacks  a  solid 
foundation  because  it  does  not  provide  for  any  of  the  links  between  the  higher 
ascomycetes  and  the  yeasts.  On  the  contrary,  Guilliermond's  theory  rests  on  a 
series  of  known  facts. 


CHAPTER  VI 

METHODS  OF  CULTURE  AND  ISOLATION  OF  YEASTS. 
PROCEDURES  FOR  THEIR  STUDY 

A.  Methods  of  Culture 

WITH  the  exception  of  a  few  pathogenic  varieties,  all  of  the 
yeasts  isolated  up  to  the  present  time,  grow  well  on  artifi- 
cial media.  They  may  be  cultivated  according  to  the  same 
methods  as  bacteria.  These  procedures  are  sufficiently  well  known  and 
are  outlined  in  detail  in  all  books  on  bacteriology.  It  would  be  out 
of  place  to  mention  them  in  a  book  of  this  nature.  It  will  suffice -to 
mention  here  a  few  of  the  media  and  methods  which  are  especially 
adapted  to  the  growth  of  yeasts.  Like  the 
bacteria  the  yeast  may  be  cultivated  as  well 
on  liquid  as  on  solid  media.  As  a  rule, 
unlike  the  bacteria,  the  yeasts  desire  a 
slightly  acid  medium.  The  yeasts,  although 
facultative  anaerobic,  multiply  only  on  media 
which  are  well  aerated.  Since  yeasts  vegetate 

more  often  at  the  bottom  of  cultures,  it  is   Fi%6£  —  A,  Pasteur  Flask; 

B,  Chamberland  Flask, 
then  necessary  to  place  them  in  thin  layers 

of  medium  in  order  to  supply  as  much  air  as  possible.  On  the  contrary, 
if  a  fermentation  is  desired,  they  should  be  placed  under  conditions 
with  a  limited  supply  of  oxygen  and  in  a  sugar  medium  contained 
in  deep  flasks  or  tubes. 

For  the  culture  of  yeasts  the  same  ordinary  apparatus  is  utilized 
as  in  the  study  of  bacteria  (Petri  dishes,  Erlenmeyer  flasks,  Roux 
tubes,  cover  glasses,  test  tubes).  For  physiological  investigations, 
Pasteur,  Chamberland,  Freudenreich  and  Hansen  flasks  are  service- 
able. The  Pasteur  flask  is  shown  in  Fig.  62.  It  is  provided  with 
two  outlet  tubes,  one  of  which  is  bent  and  which  contains  a  bit  of  cot- 
ton to  filter  the  air  which  is  thus  able  to  pass  in.  The  other  is  a  long 
straight  tube  which  enters  the  flask  at  the  side.  It  is  closed  with 
a  piece  of  rubber  tubing  carrying  a  piece  of  glass  rod  in  one  end. 
The  other  end  of  this  tube  is  slipped  over  the  tube  from  the  flask. 
Sterilization  may  be  accomplished  by  putting  in  boiling  water.  Dur- 
ing this  sterilization,  the  rubber  tube  may  be  removed  from  the 

147 


148          METHODS  OF  CULTURE  AND   ISOLATION 

straight  side  arm.  It  may  be  replaced  as  soon  as  the  operation  is 
completed.  It  is  not  necessary  to  sterilize  in  the  autoclave.  The 
Chamberland  flask  (Fig.  62)  is  an  ordinary  flask  in  which  the  col- 
lar is  drawn  out  and  ground  to  receive  tightly  a  straight  cap  which, 
in  turn,  is  drawn  out.  A  piece  of  cotton  may  be  inserted  in  this. 
The  Freudenreich  flask  is  constructed  a  little  after  the  same  fashion 
but  differs  in  that  the  body  of  the  flask  is  cylindrical  instead  of  spheri- 
cal. These  may  be  sterilized  in  the  autoclave. 

Some  of  the  common  media  which  may  be  used  in  the  cultivation 
of  the  yeasts  are  mentioned  below.1 

Pasteur's  medium: 

Distilled  water 1000       grams. 

Candied   sugar 20            " 

Ammonium  tartrate 0.1 

or,  Ammonium  carbonate 1.0        " 

Ash  of  yeasts 1.0 

This  medium  was  used  by  Pasteur  in  the  greater  part  of  his 
studies  on  alcoholic  fermentation. 

Hansen's  Medium  No.  1. 

Peptone 1        gram 

Maltose 5 

Potassium  phosphate 0.3 

Magnesium  sulfate 0.2 

Distilled  water  100.0 

Hansen's  Medium  No.  2 

Peptone - 1.0    gram 

Maltose .5.0 

Potassium  phosphate 0.3 

Magnesium  sulfate 0.5 

Distilled  water 100.0 

Mayer's  Culture  Fluid: 

Sugar 15      grams. 

Potassium  phosphate ' 5 

Magnesium  sulfate 5 

Calcium  phosphate 0.5 

Ammonium  nitrate 0 . 75 

Distilled  water 1000.00  c.c. 

According  to  Mayer,  this  is  a  very  useful  medium  for  culturing 
yeasts. 

1  Investigations  by  Wildier  in  1901,  by  Williams  in  1919  and  Bachmann  in 
1919  indicate  that  some  vitamine-like  substance  may  be  necessary  for  the  growth 
of  yeasts.  Apparently  ordinary  synthetic  media  alone  are  not  entirely  satis- 
factory for  the  culture  of  yeasts. 


METHODS  OF  CULTURE  149 

Laurent's  Medium: 

Ammonium  sulfate 4.71  grams. 

Potassium  phosphate 0. 75       " 

Magnesium  sulfate 0.1         " 

Distilled  water 1000. 00      " 

To  this  medium  any  sugar  may  be  added.     It  was  used  by  Laurent 
in  his  investigations  on  the  hydrocarbon  nutrition  studies  on  yeasts. 

Haydruck's  Medium: 

Water 2000        grams. 

Saccharose 100  " 

Asparagin 2.5         " 

Potassium  acid  phosphate 50. 00       " 

Magnesium  sulfate 17. 00       " 

Cohn's  Solution: 

Distilled  water 200  grams. 

Ammonium  tartrate 2  " 

Potassium  phosphate 2  " 

Magnesium  sulfate 1  " 

Calcium  phosphate  (dibasic) 0.1  " 

Sugar 20 

Noegeli's  Medium  (No.  3). 

Distilled  water 100       grams. 

Glucose 3 

Ammonium  tartrate 0. 04       " 

Magnesium  sulfate 0. 04      " 

Calcium  chloride 0. 02      " 

Raulin's  Medium: 

Distilled  water 1500. 00  grams. 

Candied  sugar 70. 00  " 

Ammonium  nitrate : 4. 00  " 

Tartaric  acid '. 4. 00  " 

Potassium  carbonate 0. 60  " 

Magnesium  carbonate 0. 60  " 

Ammonium  sulfate 0.25  " 

Ferric  sulfate 0. 07  " 

Zinc  sulfate 0. 07  " 

Potassium  sulfate 0. 07  " 

Potassium  silicate 0. 07  " 

This  liquid,  composed  by  Raulin  in  the  course  of  investigations 
on  the  nutrition  of  Sterigmatocystis  nigra,  makes  up  a  medium  which 
is  well  adapted  to  the  develpoment  of  fungi.  It  serves  also  for  the 
growth  of  many  fungi  and  molds.  For  the  yeasts,  however,  it  does 
not  lend  itself  as  well  since  they  seem  unable  to  grow  in  it.  For  a 
certain  few  special  yeasts  it  will  work. 


150          METHODS  OF  CULTURE  AND   ISOLATION 

Yeasts  are  easily  cultivated  on  decoctions  of  various  fruits.  Malt 
extracts  and  fruit  juices,  such  as  prune  juice  together  with  decoc- 
tions of  carrots,  potatoes,  etc.,  make  good  media.  Beer  wort  is  the 
best  medium  for  the  yeasts  and  the  one  which  is  most  utilized  for 
their  culture.  It  may  be  procured  at  breweries  or  prepared  in  the 
laboratory  after  the  following  procedure:  Soak  200  grams  of  malt, 
which  has  been  previously  pounded,  in  a  liter  of  cold  water  and  bring 
slowly  to  a  temperature  of  60°  C.  Shake  once  in  a  while,  and  after 
three-quarters  of  an  hour,  add  4  grams  of  hops.  Boil  for  about  an 
hour  and  filter.  Test  the  nitrate  for  maltose  by  means  of  Fehling's 
solutions.  The  nitrate  is  then  diluted  with  distilled  water  to  yield  a 
3  per  cent  solution  of  maltose.  The  wort  is  then  filtered  and  steri- 
lized at  115°  for  20  minutes. 

The  malt  water  is  prepared  by  soaking  100  grams  of  germinated 
barley,  which  has  been  previously  boiled,  in  a  liter  of  water.  This  is 
then  heated  to  55-58°  C.  so  that  the  amylase  is  not  destroyed.  Finally 
it  is  boiled  for  5  minutes  and  filtered  for  sterilization. 

Raisin  extract  is  easily  prepared  by  soaking  a  few  grams  of  raisins 
in  a  little  water  and  filtering.  The  filtrate  may  be  sterilized  at  150° 
for  20  minutes. 

Another  very  useful  medium  is  yeast  water  prepared  as  follows: 
100  grams  of  fresh  yeast  are  boiled,  with  shaking,  in  a  liter  of  distilled 
water.  This  is  filtered  and  sterilized.  Yeast  water  is  made  up  of  am- 
monium salts,  as  paragin  and  peptones,  which  are  ideal  substances 
for  yeast  growth.  This  liquid  by  itself  is  generally  insufficient.  In 
order  to  secure  abundant  yeast  growth,  it  is  necessary  to  add  a  sugar. 
The  decoctions  of  meat  and  various  peptone  media  are  used  for  some 
of  the  pathogenic  yeasts. 

Yeasts  grow  equally  well  on  solid  media.  It  serves  because  they 
sporulate  easily  in  it  and  because  they  exhibit  certain  macroscopic 
characteristics  which  are  used  in  their  determination.  Slants  of 
potato,  beet  and  especially  carrot  and  even  sterilized  fruit  juices  are 
excellent  media.  These  may  be  solidified  in  media.  Beer  wort  may 
also  be  used  as  a  solid  medium.  It  is  sufficient  to  mix  8  per  cent  of 
gelatin  with  it. 

Methods  for  Obtaining  Sporulation 

The  study  of  sporulation  among  yeasts  required  a  special  tech- 
nique which  might  be  outlined  at  this  time.  Special  conditions  have 
been  mentioned  above.  The  cells  should  be  well  nourished  and  young. 
It  is  necessary  that  they  have  acquired  a  sufficient  reserve  in  their 
protoplasm  to  assure  the  formation  of  ascospores.  It  is  necessary, 
then,  to  cultivate  the  yeast  which  one  wishes  to  study,  in  a  nutrient 


METHODS   FOR  OBTAINING  SPORULATION          151 

medium  for  about  48  hours  with  frequent  transfers.  For  this,  beer 
wort  is  generally  used.  It  may  be  necessary  for  the  rejuvenating 
medium  to  contain  some  special  substance  which  will  stimulate  the 
formation  of  ascospores. 

The  best  method  by  which  to  make  the  yeast  sporulate  is  to  sub- 
ject it  to  a  period  of  inanition.  Under  such  conditions  the  yeast, 
rinding  it  impossible  to  vegetate,  forms 
ascospores.  The  method  devised  by  Engel 
and  perfected  by  Hansen  is  most  satis- 
factory; it  consists  of  placing  a  block  of 
plaster  of  Paris  in  the  beer  wort.  This 
plaster  of  Paris  is  mixed  with  three  parts  "^; 

of  water   and   molded  into  a    cylinder   or  Fig   g^lc^^  Dish 
truncated  cone.     It  is  important  that  the          Used  for  Sporulation. 
surface  be  smooth. 

The  conditions  for  sporulation  have  been  mentioned  in  a  former 
chapter.  It  is  known  that  certain  factors  are  indispensable:  the  free 
access  of  air,  favorable  temperature,  a  certain  degree  of  humidity, 
a  medium  which  is  not  too  acid,  not  too  alkaline  with  favorable  con- 
centration. In  order  to  realize  these  conditions,  the  block  of  plaster 
of  Paris  is  placed  in  a  dish  in  the  bottom  of  which  is  a  little  distilled 
water.  (Fig.  63.)  Enough  water  ought  to  be  in  the  dish  so  that 
about  half  of  the  block  is  covered.  The  water 
ought  never  to  cover  the  block,  for  the  block  will 
absorb  sufficient  to  support  the  development  of  the 
yeast  which  is  placed  upon  it.  In  this  way  the 
yeast  will  find  just  those  essentials  which  are  neces- 
sary for  its  growth.  The  dish  is  closed  by  a  cover 

64, Hansen's  ^n  suc^  a  wa^  ^a^  there  is  full  circulation  of  air. 

Flask  for  Cultur-  The  apparatus,  thus  prepared,  is  sterilized  in  the 

autoclave  at  115°  C.  for  a  half  hour. 

The  rejuvenated  yeast  is  placed  on  the  block  of  plaster  of  Paris. 
This  operation  is  a  very  delicate  one.  If  the  yeast  has  been  cultured 
in  a  liquid  medium,  the  cells  may  be  filtered  out.  By  means  of  a  sterile 
platinum  wire  some  of  the  cells  are  then  transferred  to  the  surface 
of  the  block.  If  the  culture  of  yeast  is  in  gelatin,  it  may  be  trans- 
ferred directly  to  the  block.  The  dish  is  then  covered  and  put  in  the 
incubator  at  a  temperature  depending  on  the  yeast  under  examina- 
tion. It  has  been  stated  that  each  yeast  has  an  optimum  tempera- 
ture at  which  it  sporulates,  which  is  generally  between  25°  and  30°. 
At  the  end  of  thirty  hours  most  of  the  cells  will  have  sporulated. 
To  avoid  the  easy  infection  of  the  dish  by -bacteria,  Hansen  has 
devised  a  special  flask  which  has  received  the  name  of  the  '*  Hansen 


152          METHODS   OF  CULTURE  AND   ISOLATION 

flask."  (Fig.  64.)  It  is  a  cylindrical  flask  fitted  with  a  glass  top 
ground  on  with  emery.  This  is  pulled  out  and  plugged  with  a  bit  of 
cotton.  A  side  tube  is  closed  with  a  piece  of  rubber  tubing.  A  cylin- 
drical block  of  plaster  is  put  into  the  flask  about  which  is  placed 
bouillon.  The  apparatus  is  sterilized  at  115°  in  the  autoclave.  The 
yeast  is  introduced  into  the  flask  in  the  usual  manner. 

Gorodkowa  1  has  recently  proposed  a  new  method  which  has  the 
advantage  of  being  less  complicated  than  the  method  of  Engel-Han- 
sen.  It  seems  to  give  equally  good  results.  It  consists  simply  in 
inoculating  a  gelatin  mixture  with  cells  of  the  active  young  yeast. 
This  medium  is  prepared  as  follows: 

Distilled  water 100        grams. 

Gelatin 1 

Meat  bouillon 1             " 

Sodium  chloride 0.5        " 

Glucose 0. 25       " 

The  yeast  develops  quite  rapidly  after  inoculation  and  the  small 
amount  of  glucose  is  not  sufficient  to  assure  its  nutrition  for  a  long 
time.  Thus,  sporulation  is  stopped  at  the  end  of  two  or  three  days. 
This  medium  was  utilized  by  Guilliermond  for  many  yeasts  with  quite 
gratifying  results. 

Many  other  methods,  founded  on  somewhat  the  same  principles, 
have  been  perfected  to  demonstrate  ascospores.  Some  consist  of  put- 
ting the  yeast  on  blotting  paper  in  distilled  water  or  on  pure  gelatin. 
(Wasserzug.)  Yeast  water  will  also  allow  ascospore  formation  which  is 
not  sufficient  to  assure  the  nutrition  of  the  yeast.  It  soon  finds  it- 
self reduced  to  a  condition  which  makes  it  sporulate.  All  sorts  of 
liquids  placed  in  extremely  thin  layers  are  sufficient  to  make  a  yeast 
sporulate  if  the  food  is  quickly  exhausted. 

Yeasts  will  also  sporulate  on  solid  media  (nutrient  agar  or  gela- 
tin). Rees  has  shown  that  slices  of  carrot  make  a  good  substrate  for 
this  purpose.  The  great  majority  of  yeasts  form  ascopores  after 
from  6  to  8  days,  sometimes  before.  They  grow  actively  for  a  few 
days  and  then  budding  slows  up  probably  on  account  of  an  accumula- 
tion of  toxic  substances  in  the  medium  and  sporulation  begins.  This 
method  is  to  be  recommended  for  cytological  investigations,  for  it 
permits  observations  from  the  germination  of  an  ascospore  to  the 
formation  of  a  new  ascospore.  It  facilitates  the  fixations  which  are 
necessary  by  making  it  easy  to  cut  out  a  piece  of  the  carrot  on  which 
the  yeast  is  growing  and  placing  it  in  the  fixing  bath.  Guilliermond 
used  this  procedure  with  success  in  his  investigations  on  the  copula- 

1  Gorodkowa.  Ueber  das  Verfahren  rasch  die  Sporen  von  Hefepilzen  zuge- 
winnen.  Bull.  Jard.  Bot.,  Petersburg,  Vol.  1,  1908. 


PURIFICATION   AND   ISOLATION   OF   YEASTS        153 

tion  and  sporulation  of  yeasts.  It  has  the  disadvantage  of  taking 
quite  a  little  time  with  some  yeasts. 

Certain  varieties  sporulate  quickly  in  special  media.  Thus,  Klocker 
has  shown  that  Schwanniomyces  ocddentalis  produces  an  abundant 
spore  formation  on  a  decoction  of  hay  with  gelatin.  On  the  other 
hand,  Saccharomyces  Ludwigii  sporulates  in  a  short  time  in  a  5  per 
cent  solution  of  saccharose  which  is  a  poor  nutrient  for  this  yeast. 

Other  yeasts  form  ascospores  in  many  common  liquids  when  foods 
become  scarce.  In  this  category  belong  the  varieties  of  the  genera 
Willia  and  Pichia,  which  sporulate  ordinarily  in  the  pellicle  on  the 
surface  of  the  media.  The  Sch.  octosporus  is  able,  oftentimes,  to  form 
ascospores  at  the  beginning  of  fermentation. 

These  are  the  principal  methods  which  are  employed  for  this  pur- 
pose. The  method  wherein  the  plaster  block  is  used  is  satisfactory 
for  most  yeasts  and  is  very  convenient.  Certain  yeasts,  however, 
furnish  few  or  no  spores  with  this  method.  The  Schizosaccharomyces 
sporulate  very  easily  on  solid  media  (gelatin  and  carrot);  the  Zyg. 
japonicus  shows  spores  only  on  nutrient  gelatin,  on  Gorodkowa's 
medium  and  the  pellicles  of  cultures.  Zyg.  Priorianus  sporulates  on 
a  plaster  block  soaked  with  beer  wort,  on  carrot  and  on  Gorodkowa's 
medium.  Finally  certain  parasites  demand  very  special  conditions. 
S.  guttulatus,  for  example,  sporulates  only  in  the  intestines  of  ani- 
mals in  which  it  lives  as  a  parasite.  This,  however,  is  an  exceptional 
case.  Zyg.  major  sporulates  only  on  plaster  blocks  which  have  been 
moistened  with  beer  wort  and  on  gelatin  to  which  milk  has  been 
added. 

In  summarizing  this  question,  it  is  well  to  note  that  the  method 
of  Engel-Hansen  is  the  most  widely  used  and  after  that  the  proce- 
dure of  Gorodkowa.  These  yield  most  consistent  results  to  which 
every  one  has  recourse  for  careful  studies  on  sporulation. 

B.  Methods  for  Purification  and  Isolation  of  Yeasts 

The  purification  and  isolation  of  yeasts  require  delicate  tech- 
nique. In  nature,  it  happens  that  yeasts  are  not  only  mixed  with  bac- 
teria and  other  fungi  but  also  with  other  yeasts.  It  is  quite  simple 
to  separate  the  bacteria  and  molds  but  more  difficult  to  separate  the 
yeasts  from  one  another.  The  yeasts  may  possess  the  same  shape, 
which  makes  it  difficult  to  distinguish  between  them.  Thanks  to  the 
careful  investigations  of  Hansen  and  Lindner,  we  possess  today  such 
methods  that  the  isolation  may  be  made  with  fair  certainty.  Two 
methods  are  available,  the  physiological  and  the  dilution  method.  The 
latter  may  be  regarded  as  fractional  culturing. 


154          METHODS   OF   CULTURE  AND   ISOLATION 

Physiological  method:  This  procedure  rests  on  the  fact  that  the 
organisms  in  a  mixed  culture  multiply  unequally  in  the  given  medium 
and  at  the  given  temperature.  Certain  species  die  or  vegetate  slowly; 
they  are  finally  eliminated  by  the  most  vigorous  varieties.  This  is, 
then,  a  selection  by  vital  concurrence  which  results  in  the  elimina- 
tion of  certain  species  by  others.  Thus,  to  separate  bacteria  from  a 
yeast  a  small  quantity  of  acid  is  added  to  the  culture  medium  (tar- 
taric,  lactic,  hydrochloric  or  hydrofluoric).  The  bacteria  prefer  the 
alkaline  media,  while  the  yeast  finds  the  acid  most  favorable.  The 
yeast  alone  develops  in  this  medium.  If,  on  the  contrary,  one  wishes 
to  secure  the  bacteria  a  little  alkali  is  added  to  the  medium.  By 
cultivating  a  mixture  of  yeasts  in  chemically  different  media  and  at 
different  temperatures,  one  is  able  to  separate  them.  Some  will  find 
one  medium  and  temperature  more  favorable. 

This  method,  which  has  been  employed  by  many  of  the  early 
workers,  particularly  by  Pasteur  and  Cohn,  is  purely  empirical  and 
does  not  give  reliable  results.  One  may  never  exactly  know  what  to 
expect  with  it.  It  is  easy  to  suppose,  for  example,  that  one  variety 
may  be  temporarily  eliminated  for  the  time  being  by  the  development 
of  another  form  which  finds  the  conditions  more  favorable;  this  last 
variety,  however,  after  developing  rapidly,  may  finally  be  suppressed  by 
another  form  which  has  been  dormant  up  to  this  time.  Many  vari- 
eties may  develop  together  if  all  of  the  conditions  are  favorable. 
This  was  the  case  when  Pasteur  purified  brewery  yeast  by  this  method. 
He  cultivated  his  yeast  in  a  solution  containing  a  little  sugar  to  which 
was  added  a  small  amount  of  tartaric  acid.  Later  investigations  by 
Hansen  showed  that  Pasteur  had  eliminated  the  bacteria  associated 
with  the  yeasts,  but  he  had  failed  to  effect  a  separation  of  the  dif- 
ferent varieties  of  wild  yeasts,  some  of  which  caused  the  diseases  of 
beer. 

It  is,  then,  impossible  to  secure  reliable  results  by  the  physiolog- 
ical method  of  dilution.  It  is  valuable,  however,  in  starting  the 
purification  because  it  allows  the  bacteria  to  be  separated  from  the 
yeasts.  The  yeasts  m'ay  be  only  definitely  separated  by  the  proce- 
dure, which  we  shall  now  take  up. 

Dilution  Method  for  Separating  Yeasts:  This  involves  a  mix- 
ture of  the  microorganisms  which  one  wishes  to  separate  until  the 
cells  are  well  isolated. 

Lister  conceived  this  procedure  for  the  separation  of  lactic  acid 
bacteria.  He  counted  the  number  of  bacteria  in  a  drop  of  sour  milk 
under  the  microscope  and  from  this  computed  the  required  amount  of 
distilled  water  which  it  would  be  necessary  to  add  to  this  drop  so  that 
a  drop  of  the  mixture  would  contain  a  single  cell.  He  prepared  such  a 


PURIFICATION   AND   ISOLATION   OF   YEASTS        155 

dilution  and  added  five  drops  to  five  flasks  of  sterile  milk.  Some  of 
the  flasks  remained  sterile  while  others  seemed  to  possess  a  pure  cul- 
ture of  lactic  acid  bacteria.  These  probably  came  from  a  single  cell. 
Pasteur  made  the  first  application  of  this  method  to  the  purification 
of  yeasts.  He  dried  a  small  amount  of  yeast  and  after  reducing  it 
to  a  powder  mixed  it  with  plaster  of  Paris.  He  allowed  this  mix- 
ture to  fall  from  a  great  height  to  make  a  dust.  At  this  moment, 
he  opened  flasks  of  sterile  media.  Some  of  the  dust  particles  carry- 
ing yeast  cells  fell  into  the  flasks  and  in  this  way  gave  him  pure  cul- 
tures. 

The  dilution  method  is  infinitely  more  certain  than  the  physio- 
logical method.  However,  it  does  not  yield  absolutely  sure  results 
but  only  probabilities.  How  is  it  possible  to  affirm  that  a  pure  cul- 
ture secured  by  this  method  came  from  a  single  cell?  In  spite  of  its 
fundamental  imperfection,  however,  Hansen  has  devised  two  steps 
founded  on  the  same  principle,  which  allows  the  necessary  accuracy. 

These  were  perfected  in  1881  by  Hansen.1  Part  of  the  yeast 
culture  is  placed  in  a  flask  and  is  diluted  with  distilled  (sterile) 
water.  After  shaking  this  flask  the  cells  will  be  separated  uniformly 
in  the  water.  A  drop  of  this  liquid  is  taken  and  the  cells 
counted  under  the  microscope.  A  drop  may  be  placed  under  a 
cover  glass  for  examination.  Let  us  suppose  that  there  are  10  cells 
in  the  drop.  If  such  a  drop  is  put  into  a  flask  and  diluted  with  20 
c.c.  of  sterile  water,  each  cell  should  be  separated  after  shaking.  If 
one  cubic  centimeter  of  this  liquid  should  be  put  into  twenty  sterile 
flasks,  theoretically  one-half  of  the  flasks  should  show  yeast  growth. 
Practically,  the  results  are  somewhat  different,  for  it  is  improbable 
that  all  of  the  flasks  received  a  single  cell.  Hansen  overcame  this 
difficulty  by  shaking  the  flasks  vigorously  to  separate  the  cells  and 
to  distribute  them  equally  in  all  of  the  dilution  water.  The  flasks  were 
allowed  to  remain  quiet  until  the  yeast  had  developed  into  colonies. 
These  could  be  seen  on  the  bottom  of  the  flasks.  The  number  of 
colonies  which  developed  gave  some  indication  of  the  number  of  single 
cells  which  were  present. 

Hansen's  second  method  consisted  in  the  employment  of  a  solid 
medium.  Gelatin  or  agar  was  used.  He  secured  his  idea  from  Koch's 
work  which  today  is  always  used.  Koch's  procedure  involved  the 
mixing  of  a  part  of  the  culture  in  a  large  amount  of  sterile  water.  A 
drop  of  this  mixture  was  introduced  into  a  flask  of  gelatin  at  30°. 
This  was  well  shaken  to  separate  the  microorganisms  in  the  medium. 
This  gelatin  was  then  poured  out  onto  plates  of  glass  which  were  in- 

1  Hansen,  E.  C.  Chambre  humide  pour  la  culture  des  org.  microscop.  Comp. 
Rend.  lab.  de  Carlsberg,  3,  1881. 


156 


METHODS   OF  CULTURE  AND   ISOLATION 


cubated  under  -sterile  covers.  The  gelatin  solidified  and  the  cells 
which  were  contained  in  it  developed  into  colonies  where  they  lodged. 
Macroscopic  examination  of  the  color  of  the  colonies  together  with 
the  microscopic  appearance  of  the  cells  gave  assurance  that  pure  cul- 
tures had  been  obtained. 

This  method  has  been  very  well  adapted  to  the  yeasts  by  Hansen. 
He  has  replaced  the  plates  of  glass   by  moist  chambers  and   Bott- 

cher  chambers  which  permit  the 
development  of  the  colonies  to  be 
followed  by  the  microscope. 

The  ordinary  moist  chamber 
(Fig.  65)  consists  of  an  ordinary 
slide,  in  the  middle  of  which  is  a 


Fig.  65.  —  Ordinary  Moist  Chamber. 


c  — 

r                                                      -i 

depression,  covered  by  a  cover 
slip.  On  this  is  suspended  a  drop 
of  the  nutrient  solution  containing  a  few  cells.  The  cover  slip  is 
sealed  with  vaseline. 

Bottcher's  chamber,  or  the  chamber  of  Van  Tieghm  and  Lecom- 
mier,  is  made  up  of  a  glass  ring  sealed  to  the  slide  with  Canada 
balsam.  (Fig.  66.)  A  little  water  is  placed  in  this  little  chamber  formed 
by  the  ring,  to  maintain  the  proper  humidity.  On  the  top  of  this 
ring  is  placed  a  cover  slip  from  which  is  suspended  a  drop  of  solu- 
tion which  contains  the  yeast  cells.  Vaseline  is  used  to  fasten  the 
cover  slip  to  the  glass  ring.  It 
is  necessary  to  sterilize  the  ap- 
paratus before  using  by  passing 
through  the  flame.  Precautions 
must  also  be  observed  to  pre- 
vent the  ingress  of  extraneous 
microorganisms.  This  apparatus 
is  very  convenient  since  it  allows  continued  observations  of  the  cell 
for  many  days  (8  or  more). 

Hansen's  procedure  for  isolating  the  cells  is  to  place  a  drop  of 
gelatin  on  a  cover  slip  which  is  ruled  into  16  numbered  squares. 
This  is  then  placed  over  a  Bottcher  moist  chamber.  As  soon  as  the 
gelatin  has  solidified  by  cooling,  the  number  of  cells  in  it  is  counted 
with  the  aid  of  the  microscope.  This  operation  is  facilitated  by  the 
rulings  which  allow  the  enumeration  of  each  cell.  The  number  of 
squares  occupied  by  the  cells  is  determined,  after  which  the  apparatus 
is  incubated  at  25°.  By  means  of  microscopic  observations  at  regular 
intervals,  it  is  easy  to  follow  the  multiplication  of  the  cells  as  they 
increase  to  form  colonies.  Pure  cultures  may  be  secured  by  inoculat- 
ing a  flask  of  media  with  one  of  these  colonies. 


Fig.  66.  —  Bottcher's  Moist  Chamber. 

a,  Cover  glass;  b,  droplets  of  nutrient  material; 
c,  glass  ring ;  d,  water. 


PURIFICATION   AND   ISOLATION   OF  YEASTS 


157 


Lindner's  Method  for  Securing  Pure  Cultures:  Lindner  has  de- 
vised many  methods  founded  on  the  same  principle  but  much  simpler. 
One  of  these  known  as  the  drop  culture  method  consists  in  diluting 
the  yeast  until  each  drop  contains  about  a  single  cell.  Beer  wort  may 
be  used  as  the  diluent.  By  means  of  a  pipette  with  a  fine  bore, 
drops  of  this  mixture  are  placed  in  the  bottom  of  sterile  Petri  dishes. 
Each  cell  will  develop  into  a  colony.  From  these 
colonies  pure  cultures  are  obtained  by  means  of 
platinum  wires.  Transfers  are  made  into  nutrient 
media  in  order  to  get  the  cells  in  greater  quantity. 
This  procedure  is  not  as  sure  as  that  of  Hansen 
but  is  short  and  serves  in  many  investigations. 

Another  method  devised  by  Lindner  is  known 
as  the  droplet  culture  procedure.  This  is  much 
like  the  above  except  that  the  solid  medium  is 
placed  on  a  cover  slip  which  allows  continued  ob- 
servations under  the  microscope. 

Determination  of  the  Number  of  Cells  in  a  Culture  and  Study  of 
the  Multiplication  Power  of  Yeasts:  Very  often  it  is  desirable  to 
calculate  the  multiplication  power  of  yeasts  or  the  time  required  for 
the  cells  to  divide.  One  method  of  doing  this  is  by  means  of  the  hemo- 
cytometer.  This  apparatus,  devised  for  counting  the  corpuscles  in 
the  blood,  consists  of  a  glass  slide  upon  which  is  fastened  a  cylindrical 


Fig.  67. —  Mode  of 
Operation  for  Drop- 
let Cultures  accord- 
ing to  the  Method 
of  Lindner. 


Fig.  68.  —  Hemocytometer. 

or  square  glass  cover  slip  with  a  cylindrical  hole  in  the  center.  In- 
side this  hole  is  fastened  another  glass  disk  which  bears  a  ruled  area. 
When  an  accurately  ground  cover  glass  is  placed  over  the  larger  cover 
glass,  this  disk  bearing  the  ruled  portion  should  be  exactly  1  mm. 
below  the  bottom  surface  of  it.  The  ruled  area  consists  of  squares 
of  different  sizes,  depending  on  the  ruling  which  is  used.  The  value 
of  these  squares  is  usually  marked  on  the  end  of  the  slide.  While 
there  are  many  different  rulings,  the  Thoma  ruling  is  as  satisfactory 
as  any.  (Fig.  68.) 

After  the  yeast  solution  is  carefully  shaken  to  distribute  the  cells 
evenly,  a  drop  of  it  is  placed  on  the  disk  of  the  hemocytometer  and 


-. 


158 


METHODS   OF  CULTURE  AND   ISOLATION 


the  cover  glass  applied.  After  the  cells  have  settled,  they  are  counted 
and  the  number  calculated  to  the  cubic  centimeter  or  millimeter 

basis.  By  repeating  this 
operation  for  several  times, 
some  idea  may  be  secured 
with  regard  to  the  progress 
of  yeast  development. 

SI  a  tor  l  used  the  warm 
stage  to  determine  whether 
there  was  a  lag  phase  in  the 
development  of  yeast. 
(Fig.  68-A.)  By  means  of 
this  method  he  was  able  to 
follow  under  the  microscope 
the  development  of  the  yeast. 
He- pictures  graphically  the 
Fig.  68-A.  — Warm  Stage  for  Observing  the  budding  of  the  yeasts 
Development  of  Yeasts.  ,  „„  -rt\  ,  f  .  *i 

(Fig.  68-B)  and  found  that 

there  was  a  short  lag-phase  in  the  yeast  development  which  lasted  for 
two  hours,  after  which  the  cells  reproduced  at  the  usual  rate.  For  spores, 
the  lag  phase  was  longer. 


Methods  of  Studying  the 
Yeasts 

Observation    of     Develop- 
ment in  Moist  Chambers:  The 

observation  of  yeasts  presents 

no  serious  difficulties  and  the 

details  will  not  be  outlined  here.   Fig.  68-B 

The  simplest  method  is  the  one 

used  by  early  workers,  Ehrenberg,  Mitscherlich,  Kiitzing,  Schulze,  and 

Brefeld;    it  consists  in  diluting  the  culture  of  yeast  to  such  a  point 

that  each  drop  contains  only  a  few  cells.     A  drop  of  this  culture  is 

put    on    a    cover    slip  which  is    placed  over  a  moist  chamber    and 

incubated  at  25°  C.     The  various  changes  which  take  place  in  this 

drop  may  be  followed  on  the  microscope.     Observations  may  be  ex- 

tended, if  desired,  for  a  long  time. 

The  use  of  the  ordinary  moist  chamber,  and  especially  that  of 
Bottcher,  is  more  convenient.  It  makes  it  possible  to  observe  a  single 
cell  or  a  small  number  of  cells  for  a  period  of  8  days  without  danger 

1  Slator,  A.  Some  observations  on  yeast  growth.  Biochem.  Jour.  12,  No.  3, 
248-258,  1918. 


Slator's  Method  for  Observing 
Yeast  Devel°Pment  on  the  Warm  Stase- 


METHODS   OF  STUDYING  THE  YEASTS  159 

of  contamination.  The  use  of  the  ruled  cover  glass  on  which  each 
square  is  numbered,  makes  it  possible  to  follow  closely  each  stage 
in  the  development  of  the  yeast.  One  is  thus  able  to  follow  the  modi- 
fications which  occur  at  regular  intervals.  By  this  method  the 
phenomena  of  budding,  sexuality,  sporulation,  germination  of  spores, 
etc.,  may  be  watched.  When  this  apparatus  is  used  for  the  study  of 
germination  of  spores,  certain  difficulties  are  encountered.  In  a 
yeast  which  sporulates  there  are  always  a  few  cells  which  have  not 
formed  ascospores.  When  transferred  to  a  new  medium,  the  as- 
porogenous  cells  develop  more  rapidly.  Thus,  for  example,  a  dilu- 
tion of  beer  yeast  placed  in  a  Bottcher  moist  chamber  may  include 
asporogenous  cells  mixed  with  ascospores.  Their  early  development 
hinders  observation.  The  simplest  method  to  get  around  this  is  to 
kill  the  vegetative  cells  with  heat;1  the  spores  being  more  resistant 
will  pass  through  such  treatment. 

In  order  to  carry  outr  this,  one  should  proceed  as  follows :  A  portion 
of  yeast  growth  from  solid  media  (agar,  gelatin,  carrot,  etc.)  is  spread, 
by  means  of  a  spatula,  on  a  sterile  cover  slip.  This  is  then  placed 
in  an  incubator  at  55-60°  for  12  hours.  The  vegetative  cells  are  not 
able  to  withstand  this  temperature  and  only  the  ascospores  survive. 
The  yeast  is  then  moistened  with  a  drop  of  water  and  a  drop  is  placed 
in  a  Bottcher  moist  chamber.  The  vegetative  cells  will  remain  in  the 
solution  but  should  cause  no  difficulty  since  that  will  not  germinate. 
Guilliermond  found  this  method  a  convenient  one  for  studying  the 
germination  of  ascospores. 

Lindner2  extolled  very  much  a  method  which  he  devised  and  termed 
the  adhesive  culture.  This  method  simply  consists  in  applying  a  thin 
layer  of  the  yeasts  or  other  organisms  to  a  cover  slip  which  is  even- 
tually placed  in  a  humid  chamber.  Let  us  suppose  that  we  wish  to 
study  the  bacteria  in  our  saliva.  All  that  would  be  done  would  be  to 
apply  the  tongue  to  a  sterile  cover  slip.  The  organisms  remain  ad- 
herent to  the  cover  slip  and  develop  in  their  own  natural  medium. 
The  colonies  may  be  sufficiently  well  isolated  for  picking  pure  cultures 
by  means  of  a  platinum  wire.  In  this  way,  this  procedure  may  be 

1  Another  method  by  which  the  same  results  may  be  obtained  has  been  de- 
vised by  Hansen.     This  investigator  has  stated  that  the  vegetative  cells,  perhaps 
on  account  of  their  age,  are  killed  by  a  period  of  one  minute  in  absolute  or  50  per 
cent  alcohol.    The  ascospores,  in  a  state  of  maturity,  resist  the  alcohol  for  a  long 
time.     This  constitutes  a  simple  method  of  getting  rid  of  the  vegetative  cells 
when  only  ascospores  are   desired.      (Hansen,   Ueber  die  totende   Wirkung  des 
Aethylalkohols  auf  Bakterien  und  Hefen.    Cent.  Bakt.  45,  1907.) 

2  Lindner,   P.     Die  Adhasionkultur,   eine  einfache  Methode  zur  biologischen 
Analyse  von  Vegetationsgemischen  in  nattirlichen  oder  kiinstlichen  Nahrsubstraten. 
Zeitschr.  Spir.  Industrie,  No.  46  and  47,   1901. 


160          METHODS  OF  CULTURE  AND  ISOLATION 

used  for  isolating  species  or  varieties.  Another  example  would  be  the 
investigation  of  the  organisms  in  the  coating  of  decayed  grapes  in  the 
fall.  A  drop  of  sterile  water  would  have  to  be  placed  on  the  cover 
slip  and  a  grape  pressed  into  it.  This  method  is  very  convenient,  re- 
quires no  special  media  and  facilitates  microscopic  examinations  of 
the  organisms.  Such  a  method  lends  itself  to  photomicrography.  In 
fact,  Lindner  1  secured  such  illustrations  with  which  to  illustrate  his 
book  on  fermentation  microorganisms.  The  colonies  develop  very 
slowly,  which  lessens  the  danger  of  impure  cultures. 

Methods  for  Investigating  the  Cytology  of  Yeasts :  At  this  time 
we  shall  refer  to  the  technique  of  histology,  especially  those  methods 
which  are  of  much  service.  The  first  step  in  the  cytological  examina- 
tion2 of  yeasts  should  be  a  microscopical  study  of  the  cells  colored 
with  neutral  red.  To  do  this,  the  living  cells  should  be  placed  in  an 
aqueous  solution  (1-10,000).  The  protoplasm  and  the  nucleus  will 
remain  uncolored;  only  the  less  vital  parts  of  the  cell  will  fix  the  dye, 
that  is,  the  vacuole  and  the  metachromatic  corpuscles  contained  in 
it.  The  dye  will  diffuse  into  the  vacuole  and  stain  it  very  lightly. 

When  careful  observations  have  been  made  on  living  cells,  with 
and  without  coloration,  one  may  undertake  deeper  investigations  in 
cells  which  have  been  fixed  with  many  of  the  available  substances. 
The  most  convenient  procedure  for  this  is  to  cut  out  a  portion  of  the 
gelatin  or  carrot  upon  which  the  yeast  is  developing  and  place  it  in 
the  fixing  bath.  When  fixation  is  completed,  the  yeast  may  then  be 
placed  on  a  cover  slip,  upon  which  has  been  spread  a  layer  of  gelatin 
to  make  it  stick.  The  mount  may  then  be  plunged  into  a  staining  bath. 
The  fixing  and  staining  will  vary,  depending  upon  whether  one  wishes 
to  study  the  nucleus  or  the  other  contents  of  the  cell. 

For  investigations  on  the  nucleus,  Guilliermond  recommends  fixa- 
tion in  Bouin's  picroformol  solution  or  Perenyi's  solution,  which  have 
the  following  composition. 

Picroformol  solution: 

Saturated  picric  acid : 75  parts 

Glacial  acetic  acid 5     " 

Formol 20     " 

1  Lindner,   P.     Atlas   der  mikroskopischen   Grundlagen    der    Garungskunde. 
2nd  Edition,  P.  Parey,  Berlin,  1910. 

2  Guilliermond,  A.    Recherches  cytologiques  sur  les  levures  et  quelques  moisis- 
sures  a  form  levures,  these  doctorate  es  sciences  (Resume  in  Revue  generate  de 
Botan.  15,  1902).    Recherches  sur  la  germination  des  spores  et  sur  la  conjugaison 
dans  les  levures.     Rev.  generale  de   Botanique,    17,   1905.     Remarques  sur  les 
re  cents  travaux  parus  sur  la  cytologie  des  levures  et  quelques  nouvelles  observa- 
tions sur  le  groupe  de  champignons.    Cent.  Bakt.  26,  1910. 


METHODS  OF  STUDYING  THE  YEASTS  161 

Perenyi's  Fluid: 

Chromic  acid,  5  per  cent 3  parts 

[  Nitric  acid,  10  per  cent 4     " 

Alcohol,  95  per  cent 3     " 

The  period  of  fixation  ought  to  be  about  12  hours.  Finally,  stain- 
ing is  accomplished  with  Heidenhain's  ferric  hemotoxylin  (mordant- 
ing with  a  2|  per  cent  solution  of  ammoniacal  ferric  alum,  washing 
rapidly  in  water  and  staining  in  a  1  per  cent  aqueous  hematoxylin 
solution).  By  this  procedure  a  satisfactory  differentiation  of  the 
nucleus  is  obtained.  The  basophile  grains  stain  quickly  but  the  meta- 
chromatic  corpuscles  generally  do  not.  The  hematoxylin  method 
of  Delafield  gives  good  result  after  fixation  in  Bourn's  solution.  These 
methods  at  once  allow  the  differentiation  of  the  nucleus,  which  appears 
with  a  diffuse  tint,  and  the  metachromatic  granules,  which  take  on  a 
wine  color.  The  preparations  thus  prepared  ought  to  be  preserved 
in  Kayser's  gelatin-glycerol  mixture  in  preference  to  Canada  balsam 
which  always  causes  a  contraction  of  the  cells. 

Alcohol,  formalin  and  Lenhossek's  fluid  1  are  the  most  useful  fix- 
ing solutions  for  the  metachromatic  granules;  they  are  not  to  be 
recommended  so  highly  for  the  nucleus.  Unna's  polychrome  blue, 
CresyFs  blue  and  methylene  blue,  employed  in  1  per  cent  aqueous 
solutions,  allow  differential  staining  of  the  metachromatic  corpuscles 
but  generally  differentiate  the  nucleus  quite  badly.  The  preparations 
obtained  by  these  dyes  decolorize  rather  quickly  in  the  gelatin-glycerol 
solution  and  are  able  to  be  preserved  only  in  Canada  balsam. 

To  demonstrate  glycogen,  LugoFs  solution  (iodin  in  potassium 
iodide)  may  be  used.  For  fats,  Flemming's  solution  2  should  be  used. 
The  preparations  are  fixed  in  it  and  the  fat  globules  are  browned 
by  it. 

Methods  for  Determining  the  Properties  of  Yeasts  towards  Sugars: 
It  is  often  very  desirable,  especially  when  investigating  a  new  yeast, 
to  determine  its  action  towards  sugars.  The  action  of  yeasts  towards 
such  hydrocarbons  constitutes  a  very  important  step  in  their  dif- 
ferentiation. The  simplest  method  is  that  devised  by  Lindner.3  It 

1  Lenhossek's  Fixing  Fluid: 

Mercuric  chloride  (sat.  in  water) 75  volumes 

Absolute  alcohol 20 

Acetic  acid 3         " 

1  Flemming's  Solution: 

Osmic  acid,  1  per  cent  in  water 15  parts 

Crystallized  acetic  acid 1     " 

Chromic  acid,  2  per  cent  in  water 4     " 

3  Lindner,  P.  Mikroskopische  Betriebskontrolle  in  den  Garungswerben. 
Paul  Parey,  6th  Edition,  Berlin.  1909. 


162          METHODS   OF  CULTURE  AND   ISOLATION 

consists  of  filling  an  ordinary  moist  chamber  with  a  drop  of  yeast 
water  containing  the  microorganisms.  By  means  of  a  sterile  plati- 
num wire  a  little  of  the  carbohydrate  which  one  wishes  to  study,  is 
added  to  the  drop.  It  may  be  necessary  to  pulverize  the  sugar  in 
order  that,  as  far  as  possible,  equal  amounts  of  the  sugar  may  be  trans- 
ferred. The  moist  chamber,  thus  prepared,  is  covered  with  a  cover 
slip  and  placed  in  an  incubator  at  25°.  The  next  day  the  preparation 
is  examined.  If  a  fermentation  has  taken  place,  the  cover  slip  may 
be  forced  up  from  the  glass  collar  upon  which  it  rested  and  a  bubble 
of  gas  may  be  seen.  (Fig.  69.)  In  order  to  make  certain  that  the 
^T  ILL  -  ui  Th  bubble  is  made  up  of  carbon  dioxide,  in 

V//'/>/'lr^i4»£%5v  ^\  part  it  is  sufficient  to  allow  a  few  drops  of 
Fig.  69.  -Fermentation  in  an  caustic  potash  to  fall  on  the  cover  slip.  If 

Ordinary  Moist  Chamber  the  bubble  is  CO2  it  will  contract  and 

disappear.  If,  on  the  other  hand,  there  is 

no  fermentation,  the  cover  slip  will  not  have  changed  place.  It  will 
be  adherent  to  the  glass  slide. 

Bronfenbrenner  and  Schlesinger's  Method  for  Determining  the 
Action  of  Microorganisms  on  Carbohydrates :  These  investigators  l 
have  proposed  a  method  for  studying  the  action  of  bacteria  to- 
wards carbohydrates  which  may  be  of  value  for  the  yeasts.  The 
method  may  be  outlined  as  follows:  One  prepares  medium  contain- 
ing 1.5%  of  agar,  0.5%  NaCl,  and  1%  peptone.  This  mixture  is 
brought  to  boiling  and  the  reaction  not  adjusted.  At  this  point  a 
suitable  amount  of  indicator  is  added  and  the  medium  distributed 
into  small  tubes  containing  1  or  2  cc.  of  medium,  autoclaved  and  stored 
on  ice.  When  used,  the  medium  is  melted  and  to  it  is  added  0.1  or 
0.2  cc.  of  a  20%  lactose  solution.  While  hot,  this  medium  is  de- 
posited in  drops  on  the  inner  surface  of  the  bottom  of  a  sterile  Petri 
dish.  This  may  be  placed  symmetrically  by  marking  the  outside  of 
the  dish.  Each  of  these  drops  is  inoculated  from  the  suspected  col- 
onies or  material,  leaving  two  drops  uninoculated  on  each  plate  as 
controls.  After  this,  a  fresh  drop  of  the  medium  is  placed  over  the 
inoculated  drops,  giving  conditions  of  slightly  lowered  O  tension  favor- 
able to  carbohydrate  metabolism  of  bacteria.  In  order  to  prevent  the 
volatilized  acids  formed  in  some  drops  from  causing  a  color  change  in 
other  drops,  filter  paper  saturated  with  NaOH  was  placed  in  the  top  of 
the  Petri  dishes.  When  desired,  sterile  slides  with  a  concave  well  may 
be  used.  The  hollow  of  the  slide  is  filled  with  lactose  agar,  prepared  as 
outlined  above,  and  a  sterile  cover  glass  placed  over  it.  This  method 
greatly  decreases  the  length  of  time  required  for  the  formation  of  gas. 

1  Bronfenbrenner,  J.,  and  Schlesinger,  M.  J.  A  Rapid  Method  for  the  Identifica- 
tion of  Bacteria  Fermenting  Carbohydrates.  Amer.  J.  Pub.  Health,  8  (1918) ,  922-923. 


METHODS   OF  STUDYING  THE  YEASTS 


163 


Klocker' s  Method  for  Estimating  Alcohol  in  Fermented  Solutions: 
Klocker  l  has  modified  the  Pasteur  drop  reaction  for  the  determina- 
tion of  alcohol  in  fermented  solutions  and  claims  to  be  able  to  deter- 
mine the  presence  of  alcohol  in  0.002  per  cent  by  volume.  Five  cubic 
centimeters  of  the  solution  are  used  in  a  vessel  180  mm.  long  and  24 
mm.  in  diameter.  The  solution  is  slowly 
warmed  over  a  wire  gauze  by  means  of  a 
gas  flame  taking  care  to  prevent  bumping. 
Characteristic  oil  drops  accumulate  in  the 
glass  tubing,  higher  up  or  lower,  depending 
on  the  concentration  of  alcohol.  By  this 
method  it  was  demonstrated  that  small 
amounts  of  alcohol  were  formed  in  yeast 
water  on  standing.  Other  substances,  such 
as  acetone,  may  give  the  reaction  but  it  is 
necessary  that  they  be  present  in  large 
amounts.  As  a  control  iodoform  may  be 
formed  by  the  following  method:  A  small 
amount  of  sodium  carbonate  (2  grams  per 
10  c.c.  of  product)  and  iodine  (0.1  gm.)  may 
be  added  and  the  temperature  brought  to 
60°  C.  until  the  iodine  has  disappeared.  On 
cooling,  crystals  of  iodoform  will  be  formed 
which  may  be  examined  under  the  micro- 
scope. 

Determination  of  Efficiency  of  Yeast :  It 
is  often  convenient  to  have  information  con- 
cerning the  efficiency  of  yeasts  especially  if 
it  is  necessary  to  select,  from  among  a 
number  of  these  organisms,  one  which  will 
cause  the  maximum  amount  of  change  in  the 
shortest  time.  Different  methods  may  be 
used.  Any  of  the  products  of  fermentation 
may  be  measured  quantitatively.  The  alcohol 
may  be  determined  at  any  time  during  the 


Fig.  69-A.  —  Klocker  Appa- 
ratus for  Determining  the 
Presence  of  Alcohol  in 
Fermenting  Solutions. 


process  of  the  fermentation  but  has  this  disadvantage  that  consider- 
able time  would  be  used  if  many  determinations  were  necessary. 
Probably  the  most  convenient  method  which  has  been  devised  is  to 
measure  the  amount  of  carbon  dioxide  which  is  formed  and  from  this 
to  determine  the  extent  of  the  fermentation. 

This  may  be  carried  out  either  volumetrically  or  gravimetrically. 

1  Klocker,  A.    The  determination  of  traces  of  alcohol  in  fermenting  solutions. 
Cent.  Bakt.  Abt.  II,  31,  108-111;    Chem.  Absts.  6  (1912),  136. 


164 


METHODS  OF  CULTURE  AND   ISOLATION 


If  one  wishes  to  use  volumetric  methods,  an  apparatus  must  be  ar- 
ranged which  will  collect  all  of  the  gas  as  it  is  formed  during  the  fer- 
^  mentation.     Such  an  apparatus  was  arranged  by  Slator.1 

GO  An  ordinary  nitrometer  will  be  sufficient  and  should  be 

' — *  filled  with  mercury  to  prevent  absorption  of  the  gases, 
which  would  occur  if  water  or  other  liquids  were  used. 
Euler  and  Lindner2  have  described  the  Meissl  ventila- 
tion valve  (see  Fig.  69-B)  which  may  be  used  to  allow 
the  carbon  dioxide  which  is  formed  during  fermentation 
to  escape  but  which  retains  the  water.  A  small  amount 
of  concentrated  sulfunc  acid  is  put  into  the  valve  to  act 
as  an  absorbent  to  retain  moisture.  The  formation  of 

Fig   69-B  carbon  dioxide  may  be  followed  by  the  loss  in  weight  at 

Meissl     Ven-  various  intervals.    These  losses  in  weight  may  be  plotted 
for^De^ermin-  according  to  the  time  at  which  they  occurred  in  such  a 
ing   the  Fer-  way  that  the  curves  may  be  made  which  express  the 
StfrfV^rtB  fermenting  ability  of  each 
(Euler  -Lind-  yeast.    By  means  of  these, 
one    is    able   to  compare 
the  yeasts  under  examination  quickly  and 
to   determine  which   is   the  most   "effi- 
cient."   Alwood 3  has  devised  a  similar 
valve  which  is  used  in  the  same  manner 
as  the  Meissl  valve.      (See  Fig.  69-C.) 
Other  devices  may  be    resorted   to  for 
reaching  the  same  end. 

Preservation  of  Yeasts:  It  is  often 
advantageous  to  keep  yeasts  over  a  long 
period  of  time  without  having  to  transfer 
them  to  fresh  media  very  often.  Such  is 
the  case  with  laboratory  collections. 
Then,  again,  it  is  often  desirable  to 
exchange  cultures  between  laboratories  Fig.  69-C. 
and  in  many  cases  the  distance  is  great 
and  months  are  required  to  cover  it.  Explorers  have  had  need 
of  preserving  the  yeasts  which  have  been  collected  in  the  countries 
which  they  visited.  According  to  the  investigations  of  Hansen,4 

1  Slator,  A.    Jour.  Chem.  Soc.  89  (1906),  128. 

2  Euler,  H.,  and  Lindner,  P.    Chemie  der  Hefe  unter  der  alkoholischen  Garung. 
Leipzig,  1915. 

3  Alwood,  W.  B.     The  fermenting  power  of  pure  yeast  and  some  associated 
fungi.    U.  S.  Dept.  Agriculture,  Bureau  of  Chemistry,  111-1908. 

4  Hansen,   E.   C.     Recherches  sur  la    physiologic   des  ferments  alcooliques. 
Comp.  Rend,  du  lab,  de  Carlsberg,  13,  1898. 


Detail    of    Alwood 
Fermentation  Valve. 


METHODS  OF  STUDYING  THE  YEASTS         165 

the  best  method  for  preserving  yeasts  consists  in  cultivating  them 
in  a  10  per  cent  sucrose  solution  with  acid.  The  sucrose  does 
not  ferment  and  is  used  very  slowly.  Of  42  yeasts  subjected  to  this 
method  only  two  have  been  encountered  which  did  not  withstand 
such  a  solution.  S.  Ludwigii  did  not  keep  longer  than  2  years, 
often  6,  and  S.  Monacensis  has  not  survived  more  tha*n  two  or 
three  years.  The  others  have  been  kept  for  from  13  to  17  years. 
Generally  speaking,  the  method  is  a  satisfactory  one.  The  Hansen 
flask  is  generally  used.  (Fig.  70.)  Jorgensen  has  sug- 
gested a  modification  of  this  flask  which  prevents 
evaporation. 

Will *  has  proposed  another  method  which  consists 
of  drying  the  yeast  and  mixing  it  with  powdered  silica, 
plaster  of  Paris  and  carbon,  the  whole  being  dried  at 
40°  and  sealed  hermetically.  By  this  method  certain 
yeasts  have  been  kept  for  9  years;  Hansen  has  shown 

that  the  yeasts   form   ascospores   during   this   period.  „     _    H 

We  have  seen  that  desiccation  is  unfavorable  to  yeasts      sen's      Flask 
and  the  cells  form  ascospores.  *°r  Culturing 

In  other  investigations,  Will2  has  shown  that  the 
preservation  of  the  yeasts  depends  upon  three  factors,  1,  the  quantity 
of  yeast,  2,  the  composition  of  the  medium,  and  3,  the  temperature. 

1  Will,   H.     Einige  Beobachtungen  iiber  die  Lebendauer  getrockneter  Hefe. 
Zeit.  f.  d.  Ges.  Brau.,  19  and  27,  1896-1904.     Beobachtungen  an  Hefekonserva- 
tion.    Cent.  Bakt.  24,  1909. 

2  Will,  H.     Beobachtungen  iiber  Vorkommen  leben-  und  vermehrungsfahigen 
zellen  in  sehr  alten  Wiirze  Kulturen  von  untergarigen  Bierhefen.     Cent.  Bakt. 
44,  1916. 


CHAPTER    VII 

METHODS  FOR  THE  CHARACTERIZATION  AND 
IDENTIFICATION   OF  YEASTS 

NOW  that  the  methods  for  isolating  the  yeasts  have  been  outlined, 
it  is  proper  to  investigate  the  procedure  for  determining  whether 
a  yeast  which  one  has  isolated  is  a  new  variety  or  whether  it  has 
been  described  before,  and  if  it  has,  in  what  genus  it  belongs.  We 
have  not  an  easy  task  before  us.  When  discussing  the  morphology  of 
the  yeasts,  it  was  pointed  out  that  it  was  very  difficult  to  distinguish 
between  them.  Their  shapes  are  very  much  the  same,  varying  between 
a  sphere,  ellipse  and  cylinder.  With  rare  exceptions  the  form  and  struc- 
ture of  their  ascs,  and  the  appearance  of  their  ascospores  do  not 
present  specific  characters.  On  the  other  hand  the  morphological  char- 
acteristics are  not  constant  but  subject  to  variation.  The  shape  and 
dimensions  of  the  cell  vary  with  the  age,  the  physical  and  chemical 
conditions  of  the  environment.  It  is,  then,  rather  difficult  to  find 
in  the  morphology  of  the  yeast  the  differential  characteristics  which 
permit  a  close  separation  of  varieties.  It  becomes  necessary  to  search 
for  distinctive  characters  among  the  varieties.  We  shall  have  to 
look  to  the  macroscopic  appearance  on  solid  media,  to  the  appearance 
of  the  scum  on  liquid  media  in  contact  with  air,  to  the  variations  pro- 
duced by  the  action  of  various  media,  and  especially,  to  the  biochemi- 
cal characteristics  of  the  variety.  Hansen's  investigations  have  shown 
that  the  shape  of  the  cell,  the  dimensions,  and  the  appearance  are,  in 
themselves,  sufficiently  reliable  factors  for  the  identifications  of  species. 
To  him  we  owe  the  solution  of  this  question  on  specification.  To 
his  work,  we  must  look  for  a  great  number  of  characteristics  and 
a  method  for  differentiating  between  species  with  all  the  security  desir- 
able. Hansen  has  used  as  determining  characters  the  shape  and 
dimension  of  the  cell  at  different  temperatures  and  in  different  media, 
the  shape  of  the  ascospores  and  their  method  of  germination,  the 
limits  of  temperature  for  budding,  the  formation  of  a  scum,  sporula- 
tion,  macroscopic  appearances  of  the  scum  and  of  cultures,  the 
biochemical  properties  and  especially  their  action  toward  different 
carbohydrates.  Lindner  has  added  to  these  the  very  convenient 
characters  determined  from  the  "giant  colony." 

We  shall  now  take  up  in  detail  the  various  characteristics  which 

166 


VEGETATION   IN   THE  SEDIMENT  167 

are  necessary  for  the  identification  of  yeasts.  To  simplify  matters, 
let  us  suppose  that  we  have  isolated  a  yeast  which  we  wish  to  iden- 
tify, to  determine  whether  it  is  a  new  variety  or  whether  it  is  a  variety 
already  known. 

Character  of  the  Vegetation  in  the  Sediment 

The  preliminary  examination  ought  to  be  concerned  with  the 
sediment.  Finally  the  microscopic  features  of  the  cells  should  be 
investigated. 

Characteristics  of  the  Sediment:  The  microscopic  investigation 
of  the  sediment  of  yeast  growth  ought  to  give  very  useful  data.  It 
may  be  able  to  remain  distributed  through  the  medium  or  fall  to  the 
bottom.  Possibly  it  will  attach  itself  to  the  sides  of  the  culture  flask. 

Shape  and  Dimensions  of  the  Cell:  The  second  step  in  the  ex- 
amination should  be  the  microscopic  examination  of  the  cells  taken 
from  the  sediment  of  a  culture  in  carbohydrate  media.  Hansen 
recommended  for  this  study  a  young  culture  grown  at  25°  for  24  hours 
or  for  3  or  4  days  at  room  temperature.  The  dimensions  of  the  cells 
are  variable  characteristics.  Beauverie l  has  recently  applied  bio- 
metric  methods  to  the  yeast.  For  this  100  cells  are  taken  from  a  cul- 
ture and  the  measurements  plotted  against  the  corresponding  cell.  In 
this  way  a  curve  may  be  drawn  and  a  polygon  made  which  will  ex- 
press the  frequency  of  certain  sizes.  From  the  appearance  of  this 
polygon,  one  may  characterize  the  species. 

The  genus  Saccharomycodes  is  easily  distinguished  by  the  elongated 
tubular  cells  and  their  mode  of  multiplication,  which  is  intermediate 
between  budding  and  fission.  In  the  same  way,  it  is  not  difficult  to 
distinguish  a  budding  yeast  from  the  Schizosaccharomyces.  Certain 
yeasts  of  the  genera  Torulaspora  and  Debaromyces  and  many  varie- 
ties of  the  Torula  possess  a  sufficiently  characteristic  spherical  shape 
with  a  great  globule  of  fat.  Other  yeasts  are  elongated  or  cylindrical 
and  bud  at  their  extremities.  (Fig.  4.) 

Aside  from  these  yeasts  which  we  have  mentioned  and  which 
possess  some  morphological  characteristic  to  differentiate  them,  the 
great  majority  of  the  yeasts  are  not  so  characterized.  Most  of  them 
may  not  be  separated  by  some  quick  microscopic  feature.  Some  of 
them  may  be  grouped  together  by  their  shape  but  no  separation  may 
be  made  between  them  by  it.  The  microscopic  examination  of  a  yeast 
tells  us  nothing  about  its  genus  or  family. 

Optimum  Temperatures  and  Limits  for  Budding :  With  regard  to 
budding  there  are  maximum,  optimum  and  minimum  temperatures 

1  Beauverie,  J.  Les  methodes  de  la  biometrique  appliquee  a  1'etude  de  levures. 
Comp.  Rend.  Soc.  Biol.  1912. 


168        CHARACTERIZATION  AND   IDENTIFICATION 

which  do  not  coincide  with  those  for  sporulation,  scum  formation, 
and  which  serve  to  distinguish  between  the  yeasts.  These  tempera- 
ture studies  are,  then,  very  useful  in  identifying  yeasts.  Below  are 
listed  the  results  of  temperature  determinations  for  a  few  varieties 
of  yeasts  cultivated  in  beer  wort. 

Maximum  Minimum 

Name  of  Yeast  Temperature       Temperature 

°C.  °C. 

Saccharomyces  cerevisiae 40  1  to  3 

Saccharomyces  pastorianus .  .         34  0.5 

Saccharomyces  intermedium 40  0.5 

Saccharomyces  validus 39-40  0. 5 

Saccharomyces  turbidans 40  0.5 

Saccharomyces  Marxianus 46-47  0. 5 

Willia  anomala 37-38  1.5 

Saccharomyces  Ludwigii 37-38  3-1 

Johannisberg  yeast  II 37-38  0. 5 

An  inspection  of  this  table  is  very  instructive.  One  sees  that 
certain  varieties  are  able  to  live  at  high  temperatures  (S.  Marxianus 
46-47°);  others,  on  the  contrary,  are  not  able  to  bud  below  34°  C. 
It  is  also  seen  that  a  determination  of  the  temperature  limits  enables 
the  separation  between  varieties  of  the  same  shape  (S.  pastorianus 
and  Saccharomyces  intermedius) .  It  has  been  shown  that  when  yeasts 
are  cultivated  at  temperatures  approaching  their  maximum  tempera- 
tures, there  is  a  tendency  to  take  the  shortest,  or  spherical,  form 
(Hansen  and  Klocker). 

Thermal  Death  Point  Determinations 

By  this  is  meant  the  amount  of  heat  which  is  necessary  to  destroy 
the  yeast.  This  varies,  of  course,  depending  on  whether  it  is  tried 
on  vegetative  cells  or  spores.  It  has  been  stated  that  the  ascospores 
are  very  resistant,  more  so  than  the  vegetative  cells.  It  is  then 
necessary  to  determine,  for  each  variet}^  the  thermal  death  point 
for  the  spore  and  the  vegetative  cell.  The  temperatures  which  are 
thus  observed  vary  with  the  age  of  the  culture  and  the  condition  of 
the  cell.  A  great  many  factors  influence  determinations  of  the  thermal 
death  point  of  microorganisms.  Some  of  these  are  the  reaction  of  the 
medium,  the  time  of  exposure,  the  presence  of  organic  matter,  the 
presence  of  spores,  etc. 

Temperature  Limits  and  Optimum  for  Ascospore  Formation 

It  has  been  shown  in  a  preceding  chapter  that  temperature  plays 
an  important  role  in  this  phenomenon.  The  investigations  of  Hansen 


TEMPERATURE  LIMITS  AND  OPTIMUM  169 

have  shown  that  ascospore  formation  has  very  definite  limits  of  tem- 
perature, and  outside  of  these  temperatures,  none  are  formed.  Cer- 
tain temperatures  are  especially  favorable  (optimum).  For  a  given 
temperature,  situated  between  these  limits,  the  ascospores  form 
most  abundantly.  Hansen  has  also  shown  that  at  the  tempera- 
ture limits  and  the  optimum  the  duration  of  ascospores  formation 
varies  with  the  variety.  He  regards  these  as  very  important  char- 
acteristics. It  is,  then,  feasible  to  determine  for  each  variety  the  fol- 
lowing: 

1.  The  temperature  limits,  maximum  and  minimum. 

2.  The  optimum  temperature. 

3.  The  temperatures  at  which  ascospores  form  between  the  tem- 
perature limits. 

To  secure  these  characteristics  with  accuracy,  the  various  yeasts 
are  placed  in  a  carefully  controlled  incubator.  They  should  be  under 
the  same  conditions  and  on  plaster  of  Paris  blocks.  The  number  of 
hours  or  days  which  are  necessary  for  the  first  rudiments  of  ascospore 
formation  to  appear,  should  be  noticed.1 

Hansen  has  expressed  graphically  by  means  of  a  curve  the  results 
secured  with  six  varieties:  S.  Pastorianus,  cerevisiae,  ellipsoideus, 
validus,  intermedius  and  turbidans.  The  temperature  of  ascospore 
formation  was  expressed  on  the  abscissa  and  the  time  of  ascospore 
formation  on  the  ordinate.  The  curves  for  all  of  the  varieties  were 
identical.  The  curves  were  convex  towards  the  temperature  axis. 
They  are  limited  since  they  do  not  go  beyond  the  temperature  of 
ascospore  formation.  When  studied  relatively,  it  can  be  noted  that 
their  convex  parts  are  little  different;  on  the  other  hand  their  extremes 
are  quite  distinct  from  one  another.  These  are,  then,  the  temperatures 
which  it  is  important  to  note  well. 

The  time  necessary  for  ascospore  formation  in  six  varieties  under 
the  same  conditions  of  temperature  is  equally  interesting  to  consider. 
At  the  maximum  temperature  ascospore  formation  is  accomplished  in 
about  30  hours;  at  the  optimum  temperature,  there  is  little  difference 
among  the  different  varieties;  at  the  lower  temperatures,  the  dif- 
ferences are  more  and  more  striking.  Thus,  for  example,  S.  cerevisiae 
does  not  develop  ascospores  at  11.5°  until  about  10  days,  S.  inter- 
medius  at  the  end  of  77  hours,  etc. 

The  following  tables  will  indicate  the  limits  of  temperature,  the 
optimum  temperature  and  the  time  necessary  for  ascospore  formation 
of  six  varieties  studied  by  Hansen. 

1  How  one  is  to  tell  when  ascospore  formation  has  begun  is  variable.  It  will 
be  necessary  to  adopt  some  criterion  for  the  beginning  of  the  ascospores. 


170        CHARACTERIZATION  AND   IDENTIFICATION 

MAXIMUM,  MINIMUM  AND  OPTIMUM  TEMPERATURES  FOR  THE  FORMATION 
OF  ASCOPORES  OF  SlX  VARIETIES  STUDIED  BY  HANSEN 

Maximum  Optimum  Minimum 

Temperature         Temperature        Temperature 

Saccharomyces  cerevisiae 37-35  30  9-11 

"             Pastorianus 29-31.5  27.5  0.5-4 

"             intermedius 27-29  25  0.5-4 

validus 27-29  25  4.8-5 

"             ellipsoideus 30. 5-32. 5  25  4. 7-5 

turbidans 33-35  29  4-8 

TIMES  AT  WHICH  ASCOSPORES  BEGIN  TO  FORM  IN  Six  VARIETIES 
STUDIED  BY  HANSEN 

,r  ,  ,7  Maximum  Optimum  Minimum 

Name  of  Yeast  m  m  y 

Temperature       Temperature       Temperature 

Saccharomyces  cerevisiae 29  hours  20  10 

"             Pastorianus 30  24  14 

"             intermedius 34  27  17 

"             validus 35  28  9 

"             ellipsoideus 36  21  11 

"             turbidans.  .31  22  9 


Sexuality:  Morphological  Characteristics  of  the  Asc  and 
Ascospores.    Germination  of  the  Ascospores 

The  copulation  which  precedes  the  formation  of  the  asc  in  cer- 
tain yeasts,  the  morphological  characteristics  of  the  asc  and  ascospores, 
and,  finally,  the  mode  of  germination  have  great  significance  in  the 
determination  of  varieties.  Thus,  the  existence  of  a  copulation  served 
Barker  in  creating  the  genus  Zygosaccharomyces,  characterized  only 
by  their  sexuality.  With  the  exception  of  the  Schizosaccharomyces 
which  possesses  an  analogous  copulation  but  in  which  the  form  and 
mode  of  cellular  division  do  not  allow  any  confusion,  all  of  the 
yeasts  in  which  the  asc  results  from  a  copulation  fall  into  the  Zygo- 
saccharomyces. It  is  true  that  Klocker  has  discovered  a  new  variety 
from  which  he  has  made  a  new  genus,  Debaromyces  globosus  and  in 
which  he  has  observed  sexual  phenomena  of  the  same  order.  This 
variety,  however,  is  distinguished  from  all  of  the  other  yeasts  by  the 
special  shape~t>f  its  ascospores,  upon  which  Klocker  has  founded  the 
genus  Debaromyces.  Therefore  if  one  encounters  a  yeast  which  indi- 
cates sexual  processes  in  the  formation  of  the  asc,  and  if  this  yeast 
divides  by  budding  and  does  not  form  ascospores  with  a  special  form, 
it  may  be  placed  with  some  certainty  with  the  Zygosaccharomyces. 

In  Nematospora  coryli  and  Monospora  .cuspidata  the  ascs  arc 
larger  and  possess  a  more  elongated  form  than  the  vegetative  cells. 


TEMPERATURE  CONDITIONS  171 

Finally  a  fixed  number  of  ascospores  is  possible,  4  in  the  former  and 
1  in  the  latter.  Among  the  other  yeasts,  (Schwanniomyces,  Torula, 
etc.)  it  has  been  pointed  out  that  the  asc  forms  after  an  apparent 
copulation. 

Sometimes  the  shape  of  the  ascospore  is  characteristic:  hat  shaped 
in  Willia  anomala,  with  rings  in  Willia  Saturnus,  with  a  knotty  mem- 
brane in  Schwanniomyces.  Thus,  the  existence  of  sexual  phenomena 
in  the  formation  of  the  asc,  the  shape  of  the  asc  and  ascospores  are 
sufficient  to  characterize  the  Nematospora,  Monospora,  Zygosaccharo- 
myces,  Debaromyces,  Schwanniomyces  and  Willia. 

The  method  of  germination  of  the  ascospores  often  furnishes  deli- 
cate information  for  the  determination  of  species.  Hansen's  re- 
searches have  made  it  possible  to  characterize  .the  Saccharomyces,  in 
which  the  ascospores  undergo  generally  a  copulation  at  the  beginning 
of  germination,  for  they  are  connected  two  by  two  with  a  copulation 
canal.  Two  new  cells  are  formed  by  a  process  intermediate  between 
budding  and  fission. 

But  for  the  great  number  of  varieties,  notably  most  of  the  indus- 
trial yeasts,  the  shape  and  dimensions  of  the  ascospores  and  their 
germination  do  not  offer  any  distinctive  factor  which  may  be  utilized 
for  their  identification. 


Temperature  Conditions  which   Influence   Scum   Formation  and 
Microscopic  and  Macroscopic  Characteristics 

The  mode  of  formation  and  the  appearance  of  the  scums  formed 
by  different  yeasts  make  excellent  characters  upon  which  to  separate 
the  yeasts.  By  this  method,  as  has  been  discussed  in  Chapter  IV, 
two  groups  of  yeasts  may  be  distinguished.  The  yeasts  of  one  group 
form  a  scum  at  the  beginning  of  fermentation;  it  is  their  mode  of 
vegetation;  the  scums  are  well  developed,  grayish,  with  folds,  and 
usually  dry.  They  contain  air.  This  group  includes  the  genera 
Willia  and  Pichia.  For  this  group  the  temperature  limits  of  budding 
and  scum  formation  are  evidently  the  same. 

However,  many  of  the  yeasts  do  not  form  a  scum  at  temperatures 
close  to  their  temperature  limits.  It  has  been  stated  that  Klocker 
has  demonstrated  the  beneficial  effect  of  alcohol  in  the  medium  on 
scum  formation  in  the  genera  Pichia  and  Willia. 

The  yeasts  in  the  other  group  form  their  scums  very  slowly  after 
the  principal  fermentation  has  terminated.  The  scums  are  rather 
viscous,  wet,  and  do  not  contain  entrained  air.  Many  of  the  yeasts 
in  this  group  form  only  a  ring  and  some  form  neither  ring  nor  scum. 
The  character  of  the  scum  of  this  group  has  served  Hansen  for  sep- 


172        CHARACTERIZATION  AND   IDENTIFICATION 

arating  species.  Hansen  has  shown  that  scum  formation  is  related 
to  the  temperature.  Certain  temperature  limits  exist,  minimum  and 
maximum.  They  vary  with  the  species  and  are  easy  to  determine. 
When  once  determined,  they  are  very  useful  for  the  separation  of 
species. 

The  scums  have  different  macroscopic  characteristics,  depending  on 
the  variety  and  the  temperature  of  culture;  it  may  cover  the  surface 
entirely,  float  about  as  a  small  island,  or  appear  as  a  ring  around  the 
walls  of  the  container.  In  most  cases  the  cells  which  make  up  the 
scum  are  united  end  to  end  to  give  somewhat  the  appearance  of  a 
rudimentary  mycelium.  In  some  of  the  bottom  yeasts  and  the  indus- 
trial varieties,  one  may  observe  the  presence  of  durable  cells.  On  the 
scum,  then,  one  should  determine  the  temperature  limits,  the  optimum 
temperature,  the  macroscopic  appearance  of  the  scum  at  different 
temperatures  and  the  microscopic  appearance  of  the  cells  which  make 
up  this  scum  at  different  temperatures.  The  relation  of  temperature 
to  scum  formation  is  a  very  important  characteristic.  This  determina- 
tion furnishes  important  data.  The  following  tables,  prepared  from 
data  secured  by  Hansen,  are  interesting. 

TEMPERATURES  AT  WHICH  SCUM  FORMATION  TAKES  PLACE  WITH  Six 
VARIETIES  AS  DETERMINED  BY  HANSEN 

,,  ,  T,  Maximum  Optimum  Minimum 

Name  of  Yeast 

degrees  degrees 


Saccharomyces  cerevisiae.  .  . 33-34                  20-22  6-7 

Pastorianus 26-28                  26-28  3-5.8 

"             intermedium 26-28                  26-28  3-5 

"             validus. 26-23                  26-28  3-5 

"             ettipsoideus 33-34                  33-34  6-7 

turbidans 36-38                  33-34  3-5 

TIMES  AT  WHICH  SCUMS  BEGIN  TO  APPEAR  IN  THE  Six  VARIETIES 
STUDIED  BY  HANSEN 

(Time  expressed  in  days) 

,,           ,  ,,                             Maximum           Optimum  Minimum 

Name  of  Yeast  , 

degrees                degrees  degrees 

Saccharomyces  cerevisiae 9  to  18                7  to  10  2  to  3  l 

"             Pastorianus 7  to  10                 7  to  10  5  to  6 l 

"             intermedius 7  to  10                 7  to  10  5  to  6 l 

validus 7  to  10                 7  to  10  5  toe1 

"             ellipsoideus 8  to  12                8  to  12  2  to  3  * 

"             turbidans 8  to  12                3  to  4  5  to  6 l 

It    is    evident    that    Saccharomyces    ellipsoideus    is    distinguished 
from  Saccharomyces  turbidans  by  its  maximum  temperature.     On  the 

Months. 


APPEARANCES  OF  CULTURES  ON  SOLID  MEDIA     173 

other  hand,  Saccharomyces  intermedius,  validus  and  Pastorianus, 
varieties  equally  closely  related,  have  the  same  temperature  limits. 
With  regard  to  the  time  necessary  for  the  scum  to  appear,  some  equally 
interesting  differences  are  brought  out. 

Macroscopic  Appearances  of  Cultures  on  Solid  Media 1 

The  various  varieties  of  yeasts  do  not  develop  after  the  same 
manner  on  solid  media  (agar  and  gelatin).  They  offer  vegetative 
growths  which  we  may  use  as  differential  characteristics.2  Certain 
varieties  liquefy  gelatin  rapidly,  others  slowly  or  not  at  all.  This  is 
an  important  characteristic.  It  is  important  to  inoculate,  the  yeast 
into  agar,  gelatin,  carrot  or  potato,  and  examine  the 
microscopic  appearance  of  the  growth  after  the  yeast 
has  developed.  The  following  determinations  may  also 
be  made. 

Plate  Culture:  This  is  prepared  by  putting  a  little 
of  the  yeast  in  dilution  into  a  Petri  dish.    The  dish  is 
partially  filled   with    gelatin  which   serves   as  a  food.  Fig.  71.— Plate 
When  the  gelatin  has  solidified,  each  cell  will  develop  ure' 

into  a  colony  which,  for  each  yeast,  will  have  some  differential 
characteristic. 

Streak  Culture:  A  test  tube  or  Petri  dish  containing  a  solid  me- 
dium with  a  large  surface  is  streaked  with  a  little  of  the  yeast.  The 
yeast  will  develop  by  growing  along  this  line  of  inoculation. 

Stab  Cultures:  The  yeast  is  stabbed  into  a  solid  medium  by  means 
of  a  stiff  platinum  wire.  This  introduces  the  yeast  into 
an  environment  which  has  a  reduced  air  supply. 

One  may  thus  obtain  many   characteristics  which 
will  serve  in  the  differentiation  of  the  yeasts.      The 
colonies  will  possess  special  forms.    Hansen,  for  instance, 
has   shown   that   on   beer    wort   gelatin,  S.  cerevisiae, 
ellipsoideus,  Pastorianus,  validus,  and  intermedius  when 
Fig.  71- A. —  inoculated  in   streak   cultures,    present   very   different 
f *  G*  1  ?"lture  appearances  to  the  naked  eye.     The  same  was  found 
out  with  regard  to  the  stab  cultures. 

1  The  Descriptive  Chart  of  the  Society  of  American  Bacteriologists  has  been 
used    by    some  investigators  in  America  for  recording  the  salient  characters  of 
yeasts.      It  has  the  advantages  of  offering  a  uniform  method  of  procedure  and 
of  recording  concisely  in  a  small  space  the  data  for  each  yeast.     The  comparison  of 
characteristics  of  yeasts  is  thus  made  easy. 

2  According  to  the  investigations  of  Orsos,  the  form  of  the  colonies  is  a  func- 
tion of  the  elasticity  of  the  medium  upon  which  the  yeasts  are.     The  state  of 
cohesion  of  the  substrate  is  one  of  the  determining  factors  and  also,  to  a  lesser 
degree,  the  activity  of  the  yeast   (Orsos,  Die  Form,  der  tierfliegenden  Bakterien 
und  Hefencolonien.     Cent.  Bakt.  54,  1910). 


174        CHARACTERIZATION   AND   IDENTIFICATION 


Giant  Colonies 

Lindner  l  has  devised  another  method  which  yields  very  good  dif- 
ferential characteristics.  This  involves  the  growth  of  giant  colonies 
which  are  much  utilized  today  by  bacteriologists  and  mycologists. 

They  are  made  by  inoculating 
a  large  surface  of  gelatin  at  a 
single  point.  The  giant  colo- 
nies grow  steadily  until  they 
have  reached  larger  propor- 
tions than  the  ordinary  colony. 
The  inoculation  is  accom- 
plished by  placing  a  drop  of 
dilution  in  the  middle  of  a 
large  surface.  At  laboratory 
temperatures  (20°  C.)  it  will 
require  two  months  for  the 
colony  to  reach  its  large  pro- 
portions.  There  is  an 
optimum  temperature  for  each 
variety  which  permits  the 
most  characteristic  form. 
Giant  colonies,  in  each  case, 
give  a  very  different  appear- 
ance. (Fig.  72.)  However  in 


Fig.  72.  —  Appearance  of  Giant  Colonies  of 
Various  Yeasts. 

1,  S.  Pastorianus;    2,  S.  mtermedius;    3,  S.  ellipsoideus  ; 
4,  S.  turbidans;  5,  S.  validus  (after  Lindner). 


most  cases  they  merely 
furnish  characteristics  of  the 
group  and  not  specific 
characteristics.  Giant 
colonies  are  sometimes  susceptible  to  variations. 

The  types  of  the  cells  in  the  mediums  also  furnish  valuable  in- 
formation. Saccharomyces  Ludwigii,  S.  marxianus,  carlsbergensis 
and  P.  membranaefaciens  form  mycelial  filaments.  Saccharomyces 
Bailii  produce  ameboid  cells  (Fig.  31). 


Biochemical  Activity  of  Yeasts 

The  action  of  the  yeasts  towards  the  different  sugars  is  valuable 
information  in  their  differentiation.  The  method  of  Lindner,2  outlined 
above,  may  be  used.  It  should  be  determined  whether  the  yeast  de- 

1  Lindner,  P.     Das  Wachstum  der  Hefen  auf  festen  Nahrboden,  Wochenschr. 
Brau.  10,  1893. 

2  Lindner,  P.    Garversuche  auf  vershiedenen  Hefen  und  Zuckerarten.    Wochen- 
sch.  Brau.  No.  17,  1900. 


BIOCHEMICAL  ACTIVITY  OF  YEASTS  175 

composes  sugars  like  saccharose  or  maltose,  and  whether  it  ferments 
others  as  saccharose,  maltose,  galactose,  fructose,  dextrose,  lactose, 
raffinose,  melibiose,  methylglucoside,  dextrine,  inulin,  etc.  This  dif- 
ferential action  towards  the  various  carbohydrates  is  important  in 
the  determination  of  the  yeasts.  Some  will  decompose  dextrose,  others 
will  not.  The  great  majority  will  not  decompose  lactose.  Lactose- 
fermenting  yeasts  are  not  uncommon,  however.  Beijerinck1  (1889) 
found  such  a  yeast  in  the  Kefir  grain.  One  was  also  found  in  Edam 
cheese  by  the  same  author.  He  called  the  one  from  the  cheese  Sac- 
charomyces  tyrocola  and  the  one  from  the  Kefir  grains  Saccharomyces 
kefir.  Grotenfeld 2  in  the  same  year  found  a  lactose-fermenting  yeast 
in  milk.  With  regard  to  these  yeasts  being  true  saccharomyces  there 
seems  to  have  been  some  difference  of  opinion,  since  others  have  been 
unable  to  detect  the  formation  of  ascospores.  Bochicchio  3  isolated  a 
non-spore  bearing  yeast  from  Grana  cheese  which  he  named  Lacto- 
myces  inflans-caseigrana.  Freudenreich  and  Jensen  4  report  a  lactose- 
fermenting  yeast  from  Emmenthaler  cheese.  Jensen  6  later  found  two 
such  yeasts  in  butter.  Maze 6  when  studying  ten  Torulae  from  cheese 
found  only  one  which  fermented  lactose.  The  others  fermented  many 
of  the  common  carbohydrates.  Duclaux  reported  three  lactose  ferment- 
ers.  Hunter 7  isolated  such  a  yeast  from  "  foamy  "  cream  and  regarded 
it  as  the  essential  organism  for  this  abnormality.  The  thermal  death 
point  of  the  yeast  seemed  to  be  near  55°  C.  Typical  spores  were  not 
demonstrated  which  would  seem  to  exclude  it  from  classification  with 
the  Saccharomycetes.  Trehalose  is  rarely  fermented  by  the  yeasts. 
Hansen  has  been  able  to  subdivide  the  Saccharomyces  into  six  groups 
according  to  their  action  on  the  carbohydrates.  He  has  used  such 
characteristics  as  the  production  of  acetone  and  other  compounds. 

Industrial  yeasts  are  often  classified  as  bottom  or  top  yeasts. 
In  the  beer  industry,  the  top  fermentation  and  the  bottom  fermenta- 
tion are  brought  about  by  certain  variations  in  the  method  of  manu- 

1  Beijerinck,  M.  W.     Die  Lactose  ein  neues  Enzyme.     Cent.  Bakt.  Abt.  L, 
6,  44,  1889. 

2  Grotenfeld,  G.    Studien  iiber  die  Zersetzung  der  Milch.     Cent.  Bakt.  Abt. 
I,  5,  607,  1889. 

3  Bochicchio,  N.    Ueber  einen  Milchzucker  Vergarenden  und  Kaseblahungen 
hervorrufenden  neuen  Hefepilz.    Cent.  Bakt.  Abt.  I,  15,  546. 

4  Freudenreich,  ]3.  V.,   and  Jensen,  O.     Ueber    die  Einfluss    des  Naturlabes 
auf  die  Reifung  des  Emmenthalerkases.    Cent.  Bakt.  Abt.  II,  3,  544. 

5  Jensen,  O.    Studien  iiber  Ranzigwerden  der  Butter.    Cent.  Bakt.  Abt.  II, 
8,  251. 

6  Maze,  P.     Quelques  nouvelles  races  de  levures  de  lactose.    Ann.  Past.  Inst. 
17,  II. 

7  Hunter,  O.  W.     A  lactose-fermenting  yeast  producing  foamy  cream.     Jour. 
Bact.  3,  293-300,  1918. 


176        CHARACTERIZATION  AND  IDENTIFICATION 

facture.  One  class  is  able  to  live  at  higher  -temperatures  while  the 
other  demands  a  lower  temperature.  This  characteristic  does  not 
seem  to  be  constant,  for  a  top  yeast  may  transform  itself  into  a  bottom 
yeast. 

Finally,  certain  other  characteristics,  as  the  amount  of  alcohol 
produced  in  a  fermentation,  parasite  or  saprophyte,  or  pathogen, 
may  be  used.  Lindner  has  also  shown  that  one  may  use  the  nitroge- 
nous and  hydrocarbon  metabolism  properties  of  the  yeast. 

^Methods  for  the  Characterization  of  the  Torula,  Mycoderma 
and  Pathogenic  Properties 

By  summing  up  all  of  the  characters,  one  may  arrive  at  a  deter- 
mination of  a  true  yeast.  But  when  a  yeast  is  encountered  which 
does  not  form  spores  or  a  scum,  as  many  of  the  industrial  yeasts 
and  pathogens,  or  if  it  grows  on  the  surface  but  forms  no  ascospores 
as  with  the  Mycoderma,  the  determination  becomes  more  complex, 
if  not  impossible.  The  most  important  characteristics  are  the  tempera- 
ture of  scum  and  ascospore  formation.  The  biochemical  character- 
istics and  the  giant  colony  formation  remain. 

It  is  almost  impossible  to  recognize  most  of  the  pathogenic  yeasts 
described  in  the  last  few  years.  Many  of  these  varieties  need  more 
study  according  to  the  newer  methods.  Leberle  and  Will 1  have  shown 
that  many  of  the  characteristics  for  the  differentiation  of  the  Myco- 
derma and  Torula  should  be  taken  from  the  biochemical  properties 
of  the  species:  assimilation  of  various  sugars,  alcohol,  organic  acids, 
resistance  of  the  various  varieties  towards  alcohol  and  oxidations  of 
these  compounds.  Lutz  and  Guegen,2  from  their  work,  have  proposed 
another  method  for  the  determination  of  the  species  which  consists 
in  microscopic  and  macroscopic  examination  of  the  yeast  on  a  great 
many  different  media.  They  propose  to  use  the  following  media: 

I.   General  Media. 

A.  Raulin's  solution,  acid  and  neutral. 

B.  Gelatin  prepared  from  Raulin's  solution. 
II.   Nitrogenous  media  with  organic  nitrogen. 

Raulin's  solution  with  urea  in  place  of  the  ammonium  nitrate. 
III.   Media  made  up  of  different  carbohydrate   materials  and  poly- 
atomic alcohols. 

1  Will,    H.      Beitrage    zur    Kenntniss    der    Sprosspilze    ohne    Sporenbildung. 
Cent.  Bakt.  19,  1907;   221,  1908;  Beitrage  zur  Kenntniss  der  Gattung  Mycoderma 
nach  Untersuchungen  von  Hans  Leberle.     Zeit.  Brauw.  28,  1910. 

2  Lutz,  L.  and  Guegen,  F.     De  runification  des  methodes  de  cultures  des 
Mucidinees  et  des  levures.    Bull,  de  la  Soc.  de  Mycologie  de  France,  1901. 


CHARACTERIZATION  OF  THE  TORULA  177 

A.   Raulin's   solution  without    carbohydrates   to   which   various 

sugars  are  added. 
IV.    Media  containing  hydrocarbons. 

A.  Starch  or  inulin  added  to  Raulin's  solution. 
V.    Various  media. 

•  A.   Milk 

B.  Potato 

C.  Carrot 

D.  Egg  albumin 

This  method  has  not  been  used  sufficiently  to  clearly  judge  its 
value. 


CHAPTER    VIII 
VARIATION  OF  SPECIES 

THIS  is  a  rather  intricate  question  to  consider.  The  characters 
which  we  have  just  studied  may  be  utilized  for  the  determina- 
tion of  a  species  when  they  are  fixed  and  do  not  vary  within  the 
species.  This  involves  the  whole  question  of  constancy  of  characters. 
Are  such  characteristics  absolutely  constant  or  only  relatively  constant? 
To  what  extent  may  they  vary?  Do  well-determined  varieties  of 
yeasts  exist  or  may  they  change  with  the  environment?  Finally,  if  these 
variations  occur,  are  they  permanent  or  simply  transitory?  There  is 
the  important  question,  for  if  the  characteristics  upon  which  we  would  de- 
termine a  yeast  vary,  all  hope  of  differentiating  species  becomes  illusory. 

Again  it  is  Hansen  l  who  has  contributed  the  most  to  elucidate 
this  problem  by  showing  that  yeasts  may  undergo  more  or  less  im- 
portant variations;  some  permanent,  others  transitory.  In  some  cases 
changes  have  taken  place  which  have  been  of  such  nature  that  the 
yeast  has  not  returned  to  its  former  state,  even  after  attempts  cover- 
ing a  number  of  years.  Let  us  consider  some  of  these  variations  which 
permit  the  determination  of  species  and  separate  them  from  each 
other.  Some  will  be  found  to  be  more  constant;  others  variable.  We 
shall  distinguish  variations  in  shape  (morphological)  from  variations 
in  function.  In  this  category,  we  shall  separate  the  variations  which 
are  temporary  from  those  which  are  permanent.  Certain  it  is  that 
such  a  division  is  arbitrary  because  modifications  in  morphology  are 
always  accompanied  by  modifications  of  physiological  activities.  It 
is  well  to  adopt  it,  however,  for  the  convenience  of  exposition. 

Morphological  Variations :  Polymorphism :  Yeasts  are  quite  poly- 
morphic and  may  show  different  shapes  in  the  same  culture.  This 
may  depend  upon  the  conditions  which  surround  the  yeasts. 

If  one  inoculated,  for  example,  a  single  cell  into  a  nutrient  me- 
dium, it  would  be  found  that  many  different  cells  would  develop 
from  this  single  cell;  from  this,  it  is  seen  that  the  yeasts  have  no 
definitely  constant  shape.  With  Saccharomyces  cerevisiae  it  has  been 

1  Hansen,  E.  C.  Experimental  studies  on  the  variations  of  yeast  cells.  Read 
before  the  Botanical  section  of  the  British  Association.  Ipswich,  Sept.  13,  1895. 
Annals  of  Botany,  9,  1905;  Ueber  die  Variation  bei  den  Bierhefepilzen  und  bei 
anderen  Saccharomyceten.  Zeit.  Brauw.  21,  1898.  Cent.  Bakt.  Abt.  II,  4,  1898; 
Recherche  sur  la  phys.  et  la  morphologic  des  ferments  alcool.  C.  R.  des  trav.  du 
lab.  de  Carlsberg,  5,  1900. 

178 


VARIATION   OF  SPECIES  179 

shown  that  the  cells  may  pass  from  the  round  shape  to  oval  and 
even  elongated  and  curled  cells.  A  great  difference  will  also  be 
seen  in  the  dimensions  of  the  individual  cells.  In  old  cultures,  yeasts 
are  generally  smaller,  on  account  of  the  scarcity  of  food  which  does 
not  permit  the  young  cells  to  become  fully  developed.  Also,  such 
cultures  will  give  ascospores  which  germinate  into  smaller  cells.  We 
have  also  pointed  out  how  the  cells  of  S.  apiculatus  may  lose  their 
apiculate  shape  during  a  few  generations.  Hansen  has  shown  that 
the  temperature  may  play  a  role  in  influencing  the  shape.  For  ex- 
ample, in  cultivating  Saccharomyces  carlsbergensis  in  beer  wort  at  27° 
and  7°  C.  this  author  obtained  two  very  different  shapes.  Those 
which  formed  at  27°  C.  presented  a  normal  appearance;  the  others, 
formed  at  7°  C.,  were  very  curious  colonies  made  up  of  elongated  cells 
forming  a  sort  of  mycelium.  The  ascospores,  themselves,  may  pre- 
sent among  the  various  individuals  of  the  same  species  very  different 
shapes.  With  P.  membranaefadens,  for  example,  the  ascospores  are 
quite  spherical  at  times,  and  at  others,  may  be  egg-shaped. 

All  of  this  simply  points  out  that  the  cells  in  yeasts  are  not  con- 
stant in  shape  and  may,  depending  upon  the  circumstances,  take  on 
variable  forms  temporary  or  permanent.  In  a  word,  they  are  poly- 
morphic. Although  a  species  may  present  these  various  forms,  there 
usually  is  a  predominant  shape  which,  to  a  certain  degree,  is  charac- 
teristic and  may  be  regarded  as  normal  for  the  species  under  question. 
In  certain  cases,  one  may  note  the  predominance  of  abnormal  forms 
among  the  normal. 

Hansen  has  taken  two  series  of  cultures  in  which  the  cells  are 
distinctly  different  from  a  single  cell  of  S.  Carlsbergensis.  One  series 
shows  oval  or  round  cells  of  the  cerevisiae  type  while  the  other  is  of 
elongated  cells,  more  like  the  Pastorianus  type.  These  last  vegetations 
are,  then,  abnormal,  although  they  persist  through  a  series  of  cultures. 
Hansen  has  been  able  to  preserve  this  variation  for  six  months.  It 
appears,  then,  that  in  the  life  of  the  yeast  diverse  variations  may 
spring  up  which  may  endure  for  a  time  and  give  the  yeast  an  abnor- 
mal appearance.  These  are  the  sppntaneous  variations  which  occur 
without  apparent  cause  and  which  may  persist  for  a  certain  period 
of  time  and  recall  the  fluctuations  or  fluctuating  variations  which 
are  so  often  encountered  with  the  higher  plants  and  animals. 

Permanent  Variations :  Aside  from  the  temporary  variations 
there  are  permanent  ones  which  persist  through  a  number  of  genera- 
tions and  very  often  become  absolutely  constant,  creating  new 
varieties.  The  investigations  of  Lepeschkin  l  offer  examples  of  more 

1  Lepeschkin,  W.  Zur  Kenntniss  der  Ehrlichkeit  bei  der  einzelligen  Organ- 
ismen.  Cent.  Bakt.  10,  Abt.  II,  1903. 


180  VARIATION   OF  SPECIES 

constant  variation  in  the  characteristics  of  yeasts.  Guilliermond  has 
observed  in  a  young  culture  of  Schizosaccharomyces  Pombe  in  beer 
wort  a  certain  number  of  abnormal  mycelial  forms  which  suggest 
the  appearance  of  little  specks  scattered  in  the  growth  among  or- 
dinary cells.  (Fig.  73.)  These  have  been  isolated  and  obtained  in 
pure  culture  and  maintained  constantly  in  the  same  mycelial  struc- 
ture. Lepeschkin  has  also  isolated  a  similar  mycelial 
structure  which  appeared  in  the  growth  of  a  young 
culture  of  Sch.  mellacei  developing  in  glucose  yeast  water. 
(Fig.  74.)  These  mycelial  forms  in  Sch.  mellacei  are 
either  with  or  without  spores.  They  make  up,  then, 
a  constant  species  incapable  of  transforming  them- 
selves into  ordinary  cells  and  seem  to  result  from  an 

73.  — •  My-  hereditary  modification  of  the  cells.     This  transforma- 
cehal    Forma-  . 

tion   in    Sch.  tion,  caused  without  apparent  cause,  seems  to  fall  into 

Pombe    (after  ^he  category  of  de  Vries'  mutations.     In  the  sporulation 
Lepeschkin).  J 

of  yeasts  we  often  find  variations.     Yeasts  seem  to  lose 

very  easily  the  power  of  forming  ascospores  and  often  do  not  recover 
it.  Definite  asporogenic  races  of  yeasts  are  thus  formed.  Hansen 
made  the  first  observations  on  this  subject. 

In  isolating  a  large  number  of  cells  of 
S.  Ludwigii,  this  author  obtained  three  dif- 
ferent races;  one  is  marked  by  its  ability  to 
form  ascospores;  another  group  is  made  up 
of  yeasts  in  which  this  power  is  almost 
extinct;  the  last  contains  yeasts  in  which  it 
has  entirely  disappeared.  There  are,  then, 
three  races,  an  asporogenic,  a  feebly  sporogenic  Fig.  74.  —  Mycelial  Forma- 

and  a  sporogenic.     The  asporogenic  race  can      tion  in  Sch.  Mellacei  (ac- 

,   .  .  cording  to  Lepeschkin). 

be  maintained  for  a  long  time. 

On  the  other  hand,  Lindner  1  has  shown  that  when  S.  Bailii,  P. 
hyalospora  and  P.  farinosa  are  cultivated  for  a  long  time  on  must 
gelatin  they  lose  completely  their  ability  to  form  spores.  Holm  has 
reported  the  same  thing  with  cultures  of  Saccharomyces  multisporus, 
cultured  for  a  long  time  on  beer  wort  with  sucrose.  Beijerinck  2  has 
secured  similar  results  to  those  of  Hansen  with  Sch.  octosporus.  In 
cultivating  this  yeast  on  nutrient  gelatin,  this  investigator  noticed 
three  types  of  colonies;  first,  white  colonies  made  up  of  cells  which 
do  not  produce  ascospores;  secondly,  light  brown  colonies  made  up 

1  Lindner,    P.      Mikroskopische    Betriebskontrolle    in    den    Garungswerben, 
Paul  Parey,  edit.  Berlin,  6th  edition  1909. 

2  Beijerinck,    M.    W.      Weitere  '  Beobachtungen    iiber    die    Octosporushefe. 
Cent.  Bakt.  3,  1897. 


VARIATION   OF  SPECIES  181 

of  a  mixture  of  sporogenic  and  asporogenic  cells;  thirdly,  clear  brown 
colonies  made  up  of  only  asporogenic  cells.  The  asporogenic  cells 
could  be  maintained  constantly  in  this  state.  Beijerinck  effected  a 
separation  of  the  types  by  heating  at  56°.  The  asporogenic  type  was 
killed  by  this  treatment,  only  the  spores  passing  through.  These 
when  grown  on  gelatin  produced  sporogenic  cells  with  only  about  1 
per  cent  of  asporogenic  cells.  These  latter  cells  increase  in  propor- 
tion as  the  cultures  are  kept  in  the  laboratory.  The  yeast  slowly 
changes  into  an  asporogenic  type. 

Both  of  these  types  present  different  physiological  and  morpho- 
logical characteristics.  The  sporogenic  type  is  made  up  of  cells 
more  elongated,  and  liquefies  gelatin  more  quickly  than  the  asporo- 
genic variety,  which  presents  round  cells  grouped  like  the  Sarcina. 
Both  varieties  may  be  recognized  macroscopically  when  treated  with 
iodine.  The  colonies  made  up  of  sporogenic  cells  are  blued  since  the 
membranes  of  the  ascospores  are  impregnated  with  starch  while  the 
asporogenic  colonies  are  colored  yellow.  This  is,  then,  a  very  definite 
example  of  a  transformation  of  a  yeast  to  a  permanent  asporogenic 
type. 

Similar  results  have  been  secured  by  Beijerinck  (?)  with  Sch. 
Pombe.  By  cultivating  this  yeast  on  nutrient  gelatin,  this  investi- 
gator noticed  the  formation  of  two  kinds  of  colonies,  one  white  and 
composed  of  sporogenic  cells,  the  other  brown,  and  made  up  of 
asporogenic  cells.  Here,  then,  we  have  both  the  sporogenic  and 
asporogenic  cells.  The  loss  of  sporulation  may  be  accompanied  by 
a  loss  of  sexuality  as  has  very  often  been  noticed  in  the  yeasts  and 
about  which  a  little  has  been  said  in  a  preceding  chapter.  This 
is  true  in  a  yeast  secured  from  Beijerinck's  laboratory  under  the  name 
of  Sch.  mellacei,  in  which  the  ascs  form  from  ordinary  cells  without 
undergoing  any  copulation.  This  yeast,  which  differs  a  little  from 
Sch.  mellacei,  seems  to  be  a  parthogenetic  variety.  Quite  a  similar 
observation  has  been  reported  with  S.  Ludwigii.  Guilliermond  had 
the  opportunity  to  observe  two  types  of  this  yeast  from  the  same 
source.  Both  came  from  Hansen's  laboratory.  With  one  the  asco- 
spores underwent  a  copulation  at  the  moment  of  germination  as  is 
the  normal  procedure  for  this  species.  In  the  other,  the  copulation 
had  entirely  disappeared.  All  of  the  variations  of  sporogenic  func- 
tion and  sexuality  which  we  have  mentioned  up  to  this  point  super- 
vene without  apparent  cause  and  may  be  regarded  as  true  mutations. 

Other  investigations  by  Hansen  on  the  loss  of  sporulation  give  us 
an  example  of  variation  produced  by  an  accurately  determined  cause. 
It  is  known  that  the  maximum  temperature  of  budding  in  a  variety 
of  yeasts,  is  always  a  few  degrees  higher  than  the  maximum  tempera- 


182  VARIATION  OF  SPECIES 

ture  of  sporulation.  Inversely  the  minimum  temperature  of  budding 
is  always  a  little  lower  than  that  for  sporulation.  What  happens, 
then,  if  one  allows  the  yeasts  to  remain  for  a  period  between  these 
two  temperatures?  Such  is  the  question  that  Hansen  tried  to  solve. 
He  obtained  a  complete  loss  of  the  ability  to  sporulate  by  cultivating 
a  number  of  the  yeasts  for  generations  in  beer  wort  at  a  temperature 
higher  than  the  maximum  for  sporulation.  He  could  not  obtain  the 
same  results  by  placing  the  yeast  at  a  temperature  lower  than  the 
minimum  for  sporulation.  The  transformation  is  accomplished  slowly 
and  by  successive  culturing;  the  number  of  sporogenic  cells  gradually 
diminish  until  they  totally  disappear.  Thus  may  be  obtained  as- 
porogenic  varieties  which  may  be  maintained  indefinitely.  Hansen 
has  been  able  to  keep  them  for  sixteen  years  without  taking  up  the 
sporogenic  property  again.  The  types  of  yeasts  thus  secured  may 
be  regarded  as  constant.  These  varieties  offer  new  characteristics 
which  give  evidence  of  profound  modifications  in  the  structure  of  their 
protoplasm.  The  power  of  budding  often  increases  and  the  colonies 
present  a  different  appearance  than  yeasts.  On  the  other  hand,  all 
of  the  varieties  thus  obtained,  with  certain  rare  exceptions,  seldom 
produce  a  scum.  Thus  the  loss  of  power  to  form  spores  is  a  charac- 
teristic definitely  acquired  by  this  variety  when  cultivated  for  a 
certain  time  on  beer  wort  at  a  maximum  temperature  for  the  form- 
ation of  endospores. 

This  transformation  of  a  species  which  is  sporogenic  into  an  as- 
porogenic  type  is  a  most  typical  example  of  an  acquired  charac- 
teristic which  may  approach  the  attenuation  of  a  virus,  as  shown  by 
Pasteur.  How  Pasteur  attenuated  the  Bacillus  anthracis  by  grow- 
ing it  at  a  temperature  of  42-43°  C.  is  well  known.  Not  only  the 
toxic  properties  of  the  bacillus  disappeared  but  also  its  sporogenic 
functions.  Here  we  have  the  creation  of  a  new  type  characterized 
by  the  loss  of  virulence  and  ability  to  form  spores.1 

How  does  this  loss  of  ability  to  sporulate  in  yeasts  operate?  Is 
it  a  transformation  or  a  selection?  Hansen  does  not  regard  it  as  a 
selection  because  many  of  the  cultures  of  yeasts  which  he  used  to 
produce  this  spprogenic  type,  especially  the  yeast  Johannisberg  II, 
contains  only  sporogenic  cells.  Numerous  observations  have  convinced 
him  that  an  asporogenic  cell  exists  in  this  culture.  It  must  be  a  typi- 
cal transformation. 

Hansen  has  shown  that  by  varying  the  composition  of  the  medium 

1  Hansen  has  approached  this  transformation  of  a  sporogenic  into  an  asporo- 
genic yeast  by  certain  variations  which  have  been  observed  in  the  higher  plants. 
In  America  the  banana  reproduces  asexually  while  in  mid-Asia  it  reproduces 
sexually. 


VARIATION  OF. SPECIES  183 

and,  for  example,  employing  a  solution  made  up  of  peptone,  maltose, 
and  various  salts  or  a  must  gelatin,  that  the  composition  of  the 
medium  does  not  play  any  r61e  in  this  transformation.  The  same 
is  true  for  aeration  of  the  culture.  The  only  factor  which  seems  to 
have  any  effect  is  the  temperature. 

The  formation  of  sporogenic  and  asporogenic  types  of  yeast  in  the 
same  culture  of  yeast  seems  rather  common.  Nadson,  rather  recently, 
has  observed  asporogenic  varieties  in  Nadsonia  fulvescens.  The  col- 
onies of  this  type  have  a  white  color  which  distinguishes  them  from 
the  sporogenic  colonies  which  are  reddish. 

Saito  has  noticed  in  Zygosaccharomyces  Mandshuricus  the  forma- 
tion of  asporogenic  types,  indicated  by  a  transparent  yellow  color,  while 
the  sporogenic  types  have  a  white  color.  The  asporogenic  type  ap- 
pears as  a  mutation.  If  the  white  colonies  are  isolated  both  sporo- 
genic and  asporogenic  colonies  are  obtained.  When  the  asporogenic 
colonies  are  isolated,  one  obtains,  almost  exclusively,  asporo- 
genic yellow  colonies.  There  seems  to  be  a  tendency  to  return  to  the 
sporogenic  type  as  is  shown  by  other  data.  The  sporogenic  type 
is  distinguished  from  the  asporogenic  type  by  a  certain  number  of 
characteristics.  The  asporogenic  type  contains  but  a  small  amount 
of  glycogen.  Their  reaction  towards  Lugol's  iodine  allows  them  to  be 
distinguished  macroscopically.  On  the  other  hand  the  asporogenic 
variety  liquefies  gelatin  while  the  sporogenic  race  does  not.  The 
sporogenic  type  forms  a  deposit  of  spherical  cells  at  from  28°  to 
30°  while  the  other  type  forms  long  cells  sometimes  in  chains. 

This  ic  contrary  to  the  observations  of  Hansen  on  S.  pastorianus 
and  the  yeast  Johannisberg  II,  in  which  the  sporogenic  race  was  only 
slightly  formed;  in  S.  mandshuricus,  as  in  Sch.  octosporus  and  Nad- 
sonia, the  asporogenic  varieties  appeared  quickly  and  do  not  seem  to 
depend  on  conditions  of  culturing  but  on  internal  conditions.  A  low 
temperature,  however,  as  in  Schizosaccharomyces  octosporus.,  favors  the 
formation  of  asporogenic  races,  and  in  old  cultures  the  asporogenic 
types  seem  to  predominate. 

Saito  has  also  observed  the  formation  of  asporogenic  races  in 
Zygosaccharomyces  Mandshuricus.  There  are  white  colonies  and  gray- 
ish yellow  colonies  with  irregular  surfaces.  The  inoculation  of  a  white 
colony  produces  a  majority  of  white  colonies  with  a  few  yellow 
colonies.  The  inoculation  of  grayish  yellow  colonies  gives  the  asporo- 
genic cells.  The  asporogenic  race  is  distinguished  from  the  sporo- 
genic race  by  the  shape  of  its  cells,  longer  and  arranged  in  chains 
with  less  glycogen.  The  white  race  which  is  not  definitely  asporogenic 
has  a  tendency  to  lose  its  sexuality  and  to  give  parthenogenetic  ascs 
after  unfruitful  attempts  at  copulation. 


184  VARIATION  OF  SPECIES 

Physiological  Variations:  Besides  morphological  variations,  one 
may  also  observe  physiological  variations.  A  yeast  may,  for  example, 
under  certain  conditions,  induce  more  or  less  active  fermentations  in 
the  same  way  that  a  bacterium  may  be  made  more  or  less  virulent. 
But  while  certain  bacteria,  Bacillus  anthrads  for  instance,  may  be 
made  avirulent,  among  the  yeasts  it  is  impossible  to  suppress  the  fer- 
menting function.  One  may  decrease  it  or  even  increase  it  but  not 
entirely  blot  it  out. 

The  first  investigations  on  variation  of  physiological  nature  in 
yeasts  were  carried  out  by  Hansen.  When  cultivating  two  races  of 
Saccharomyces  carlbergensis  for  a  long  time  in  two  series,  one  on  ordinary 
beer  wort  and  the  other  on  the  same  substance  to  which  gelatin  had 
been  added,  he  was  able  to  build  up  a  more  actively  fermenting  type 
on  the  gelatin  medium.  By  cultivating  the  ascospores  of  Saccharo- 
myces cerevisiae  in  gelatin  with  yeast  water,  the  same  investigator  ob- 
tained a  variety  which  would  form  from  one  to  three  per  cent  more 
alcohol  than  the  original  culture.  On  the  other  hand,  by  cultivating 
Saccharomyces  carlbergensis  in  must  at  32°  C.  Hansen  has  obtained  a 
variety  which  formed  less  alcohol  than  the  normal.  According  to 
Hansen,  these  results  are  due  to  a  selection  born  of  a  transformation. 
The  most  active  type  will  tend  to  be  built  up.  From  all  of  these 
examples  which  we  have  mentioned  one  is  justified  in  concluding  that 
cells  of  the  same  species  of  yeast  often  present  great  differences  and 
that  new  varieties  may  be  created  by  selection  which  have  special 
physiological  properties. 

This  is  the  point  of  departure  from  the  use  in  the  industries  of 
"  selected  yeasts."  By  making  a  series  of  cultures  from  a  single  cell, 
as  each  cell  possesses  slightly  different  physiological  properties,  one 
may  obtain  strains  presenting  the  definite  properties  of  the  original 
cell.  Some  will  be  more  feeble  and  others  more  active.  The  latter 
have  been  termed  "  yeasts  by  selection  "  for  they  may  be  maintained 
for  a  longer  or  shorter  period  and  are  then  able  to  yield  the  best 
results  in  the  industries. 

All  of  the  physiological  variations  which  we  have  just  mentioned, 
increase  or  decrease  of  the  fermenting  function,  are  abrupt  transfor- 
mations. It  is  now  time  to  look  into  the  work  of  Effront  and  other 
workers  for  examples  of  transformations  due  to  determined  causes, 
as  the  becoming  accustomed  to  chemicals. 

The  work  of  Beinarcki  has  shown  that  the  antiseptics  in  small 
doses  progressively  increase  the  fermenting  power  of  yeast  up  to  a 
certain  limit  where  the  yeast  degenerates  and  dies.  There  are,  then, 
doses  which  "  favor  "  this  ability  up  to  certain  limits.  Effront l  has 

1  Effront,  J.  L'influence  de  1'acide  fluorhydrique  et  des  fluorures  sur  lea 
levures  de  biere.  Comp.  Rend.  Acad.  Sciences,  117,  1893;  118  and  119,  1894. 


VARIATION  OF   SPECIES  185 

studied  the  effect  of  fluorides  and  hydrofluoric  acid  on  yeasts.  A 
special  concentration  exists  where  the  vegetative  growth  is  greatest, 
and  also  where  the  fermenting  activity  is  greatest.  These  two  optima 
do  not  coincide.  When  the  fermenting  power  is  greatest,  the  vegeta- 
tion is  absent.  Dienert,1  in  about  the  same  manner,  has  shown  that 
when  yeasts  which  ferment  galactose  actively  are  placed  in  a  saccharose 
solution  and  eventually,  after  washing,  are  placed  in  galactose,  they  fail 
to  induce  a  fermentation  quickly.  Only  after  from  24  to  36  hours  is 
a  typical  fermentation  started.  If  the  same  experiment  is  repeated 
with  the  exception  that  galactose  replaces  the  saccharose,  the  fer- 
mentation will  start  in  about  an  hour.  The  time  for  fermentation  to 
start  is  thus  greatly  shortened.  By  the  latter  treatment,  the  yeast 
has  been  "  accustomed  "  to  the  galactose  by  the  preliminary  treat- 
ment. 

These  results  compare  with  investigations  of  Duborg.2  It  is  known 
that  the  greater  number  of  the  yeasts  are  able  to  ferment  galactose. 
Duborg  has  been  able  to  train  yeasts,  which  normally  do  not  ferment 
this  sugar,  to  ferment  it.  He  cultivated  his  yeasts  in  a  liquid  very 
rich  in  carbohydrate  materials  (yeast  water,  25  per  cent,  glucose  5 
per  cent  and  galactose  5  per  cent).  It  is  the  cultivation  of  the  yeast 
in  this  solution  in  the  presence  of  galactose  which  gives  it  a  power 
which  it  did  not  possess.  The  more  recent  investigations  of  Harden 
and  Norris3  have  confirmed  these  data. 

An  increase  in  the  activity  of  zymase  may  also  be  explained  by 
these  data.  Duborg  has  gone  still  further.  He  claims  that  a  yeast 
which  does  not  invert  cane  sugar  may  be  made  to  do  so  by  cultivating 
it  in  a  nitrogenous  medium  containing  dextrose  and  saccharose.  This 
statement  has  been  refuted  by  Klocker  and  Hansen  who  claim  that  a 
yeast  which  does  not  ordinarily  decompose  sucrose  cannot  be  made 
to  do  it.  According  to  these  authors  4-5  the  possession  of  invertase  is 
a  constant  characteristic  and  useful  in  the  determination  of  the  yeasts. 

Other  investigations  on  physiological  variations  have  been  carried 

1  Dienert,   G.     Sur    la  fermentation  de  la  galactose    et   1'accoutumance  des 
levures  a  ce  sucre.    Ann.  Inst.  Past.  14,  1900. 

2  Duborg,    E.      De   la   fermentation   des   Saccharides.      Comp.    Rend.    Acad. 
des  Sciences,  128,  1899. 

3  Harden,  A.  and  Norris,  R.     The  fermentation  of  galactose  by  yeast  and 
yeast  juice.    Proceedings  of  the  Royal  Society,  82,  1910. 

4  Klocker,  A.     La  formation  des  enzymes  dans  les  ferments  alcooliques  peut- 
elle  servir  a  caracteriser  1'espece?    Comp.  Rend.  lab.  de  Carlsberg,  50,  1909. 

5  Hansen,  E.  C.    Recherches  sur  la  physiologic  et  la  morphologic  des  ferments 
alcooliques.     XI.     La  spore   de  saccharomyces  devenue  sporange.     Recherches 
comparatives  sur  les  conditions  de  vegetative  croissance  et  le  developpement  des 
organes  de  reproduction  des  levures  et  des  moisissures.     Comp.  Rend,  du  lab. 
de  Carlsberg,  5,  Book  2,  1902. 


186  VARIATION  OF  SPECIES 

out  by  Will 1  and  Jorgensen.2  Under  industrial  conditions,  degenera- 
tions of  yeasts  which  have  been  pure  when  used  have  often  been 
obtained.  A  yeast  which  may  always  have  given  good  results 
may,  of  a  sudden,  give  a  beer  with  evident  defects.  The  fermentation 
is  too  slow  or  too  rapid,  or  the  beer  takes  on  an  abnormal  taste.  A 
degeneration  of  the  yeast  has  taken  place.  Will  has  noticed  that  this 
may  occur  in  the  cells  of  scum  yeasts  more  than  in  those  of  bottom 
yeasts.  According  to  Will,  the  yeast  may  be  regenerated  by  repeated 
culturing.  On  the  other  hand  Jorgensen  has  arrived  at  similar  con- 
clusions. It  seems  then,  that  the  cells  in  scum  yeasts  are  more  liable 
to  degeneration. 

We  shall  now  consider  the  results  secured  by  Hansen3  on  the 
transformation  of  bottom  yeasts  into  top  yeasts.  It  has  been  pointed 
out  that,  from  the  industrial  viewpoint,  yeasts  are  divided  into  two 
groups,  bottom  and  top.  The  first  are  those  which  only  produce  fer- 
mentations at  the  higher  temperatures,  the  second  class  produce 
fermentations  at  the  lower  temperatures.  Hansen  had  noticed  that 
certain  yeasts  of  the  bottom  type,  after  having  been  cultivated  for 
a  period  of  time  at  low  temperatures  are  able  to  induce  top  fermenta- 
tions. He  then  searched  for  an  explanation  of  this  observation, 
taking  Saccharomyces  turbidans  which  is  well  known  as  a  bottom  yeast. 

He  inoculated  a  trace  of  this  yeast  into  flasks  containing  beer 
wort  and  left  them  for  from  3  to  5  months  at  a  temperature  of  5°  C. 
after  which  he  transferred  some  cells  from  these  flasks  to  others  at 
more  favorable  temperatures.  The  yeast  thus  obtained  produced 
a  top  fermentation.  Of  130  cells  which  he  examined,  none  produced  a 
bottom  fermentation.  Hansen  has  thus  obtained  the  transformation 
of  a  yeast,  which  is  normally  a  bottom  yeast  into  a  top  yeast  by  simply 
keeping  it  at  a  temperature  of  5°  C.  What  is  the  cause  of  this  trans- 
formation? In  order  to  seek  an  explanation,  Hansen  analyzed  the 
properties  of  the  cells  of  a  culture  of  Saccharomyces  turbidans  which 
were  subject  to  this  transformation.  He  examined  100  cells  and  found 
that  one-half  offered  characteristics  of  a  top  yeast  and  one-half  had 
the  characteristics  of  a  bottom  yeast.  He  extended  his  observations 
by  inoculating  cells  from  mixed  industrial  yeasts  into  separate  flasks 
and  incubating  at  5°  C.  At  the  end  of  from  3  to  4  months,  the  cells 
of  the  bottom  yeasts  had  given  no  growth  while,  on  the  other  hand, 
the  cells  from  top  yeasts  had  given  evidence  of  development.  The 

1  Will,  H.    Zeitschr.  f.  d.  ges.  Brau.  21,  1898. 

2  Jorgensen,  A.     Untersuchungen  iiber  das  Ausarten  der  Brauereihefe.     Zeit. 
f.  d.  ges.  Brau.  21,  1898.    ' 

3  Hansen,  E.  C.     Oberhefe  und  Unterhefe.     Studien  iiber  Variation.     Cent. 
Bakt.  15,  1905;    18,  1907. 


VARIATION  OF  SPECIES  187 

cells  of  the  bottom  yeast  continued  to  give  a  bottom  fermentation 
and  those  of  top  yeasts  a  top  fermentation.  This  seemed  to  show 
that  there  was  no  transformation  from  a  bottom  yeast  into  a  top 
yeast  but  probably  a  selection;  at  a  temperature  of  5°  C.,  the  cells 
from  the  top  yeast  developed  alone  while  the  cells  of  the  bottom 
yeast  remained  stationary.  Their  properties  however  were  not 
modified.  Saccharomyces  turbidans  offers,  then,  as  Hansen  de- 
scribed in  1883,  characteristics  of  a  bottom  yeast.  Such  changes 
have  been  induced  since  that  time  that  part  of  the  cells  in  a  culture 
cause  a  bottom  fermentation  and  part  a  top  fermentation.  This 
change  had  been  induced  spontaneously  while  the  yeast  was  in  Han- 
sen's  laboratory  and  without  apparent  cause.  Furthermore  this 
characteristic  seems  to  have  been  retained,  since  Hansen,  on  later 
observations,  found  the  same  proportion  of  cells  of  both  types  of 
yeast.  This  phenomenon  is  related  to  the  mutations  of  de  Vries. 

Studies  on  other  bottom  yeasts  by  Hansen  have  also  given  some 
evidence  of  a  change  from  bottom  to  top  yeast.  He  isolated  1000 
cells  from  the  yeast  Johannisberg  77,  which,  through  a  number  of 
generations,  had  given  a  bottom  fermentation.  He  cultivated  these 
separately;  984  gave  a  true  bottom  fermentation  while  16  produced 
an  intermediary  fermentation.  The  cells  inducing  bottom  fermenta- 
tion tended  to  remain  constant  while  those  in  the  top  fermentations 
tended  to  be  changed. 

Two  of  the  16  cultures  which  induced  an  intermediary  fermenta- 
tion, were  studied  further.  Here  are  the  results  for  one  of  them:  of 
100  cells,  5  gave  a  top  fermentation,  55  an  intermediary  fermentation 
and  40  a  bottom  fermentation.  Other  observations  were  made  on  the 
5  cells  which  produced  the  top  fermentation.  Of  100  cells,  78  gave  a 
top  fermentation,  9  an  intermediary  fermentation  and  13  a  bottom 
fermentation.  In  this  particular  case  there  was  a  more  or  less  definite 
movement  toward  the  top  fermentation.  Similar  results  have  been 
obtained  by  using  ascospores  in  place  of  the  vegetative  cells. 

Saccharomyces  carlbergensis  and  monascencis  gave  similar  results. 

On  the  contrary,  Hansen  did  not  observe  this  tendency  on  the  part 
of  the  top  yeasts  to  transform  into  bottom  yeasts.  They  seemed  to  be 
much  more  stable.  He  was  not  able  to  transform  Saccharomyces  vali- 
dus,  a  typical  top  yeast,  into  one  which  would  produce  a  bottom  fer- 
mentation. In  one  analysis,  he  could  find  only  3  "  bottom  cells  "  in 
100  and  he- could  not  increase  this  number.  In  another  analysis,  out 
of  1529  cells  only  one  was  separated  which  induced  bottom  fermenta- 
tion. With  Saccharomyces  cerevisiae,  another  top  yeast,  out  of  2423 
cells,  Hansen  found  only  7  which  gave  a  bottom  fermentation  and  a 
more  careful  study  of  the  vegetation  formed  by  these  indicated  that 


188  VARIATION  OF  SPECIES  < 

they  were  mixed  types  which  only  tended  very  slightly  to  induce 
bottom  fermentation. 

These  experiments  show  that  the  distinction  set  up  between  top  and 
bottom  yeasts  does  not  exist;  in  reality,  one  may  find  both  cells  of 
top  and  bottom  yeasts  in  the  same  culture.  It  may  be  that  the 
conditions  in  the  environment  favor  the  development  of  one  type,  as 
with  Saccharomyces  turbidans  incubated  at  5°  C.  The  bottom  yeast 
may  thus  change  into  a  top  yeast,  and  perhaps  top  yeasts  into  bottom 
yeasts. 

In  summarizing,  it  is  apparent  that  the  same  species  of  yeast  may 
undergo  important  variations  in  morphology  and  function.  Thus,  a 
new  series  of  varieties  and  types  may  be  created  in  which  particular 
characteristics  are  maintained  for  a  certain  time,  be  it  indefinitely. 
Thus  also,  with  regard  to  physiological  functions,  the  fermenting  func- 
tion is  susceptible  in  a  certain  measure;  of  being  enfeebled  or  increased, 
or  a  top  yeast  may  change  into  a  bottom  yeast.  Is  one  justified,  in 
light  of  these  data,  in  refusing  to  differentiate  between  species?  Cer- 
tainly not.  We  may  use  the  temperature  limits  for  the  formation  of 
ascospores,  scum  and  for  budding,  and  the  action  toward  different 
sugars.  The  characteristics  of  the  ascospores  and  germination  main- 
tain themselves  without  undergoing  modification.  We  may  conclude 
that  if  the  species  is  not  definitely  constant  in  yeast,  it  is  as  constant 
as  with  the  other  plants.  More  difficulties  are  encountered  with  the 
yeasts  on  account  of  the  variability  of  a  great  number  of  character- 
istics. 

"All  of  the  variations  of  species  seem  to  be  more  doubted  than 
with  the  higher  plants;  variations  are  more  rapid  with  the  yeasts. 
Further,  the  specific  characters  are  less  definite  than  in  the  higher 
plants  and  one  may  distinguish  less  easily  those  which  are  constant 
and  specific  from  those  which  are  not.  One  may  encounter  yeasts 
scarcely  more  variable  than  the  higher  plants,  but  their  generation 
time  is  much  shorter  and,  consequently,  the  phenomena  of  variation 
appear  more  quickly.  Here  the  investigator  may  be  witness  to  re- 
markable transformations  in  a  short  time."  Such  are  the  words  which 
Hansen  has  used  with  regard  to  this  question. 


CHAPTER   IX 
CLASSIFICATION  OF  THE  YEASTS 

WE  have  seen,  in  the  preceding  chapters,  that  the  yeasts  may 
be  regarded  as  making  up  a  group  of  lower  Ascomycetes 
closely  related  to  the  family  of  Endomyces.  It  has  been 
shown,  also,  that  they  seem  to  be  derived  from  an  ancestral  form  re- 
lated to  Eremascus  fertilis  which  may  give  birth  at  times  to  various 
representatives  of  Endomycetes  and  yeasts.  On  account  of  the  close 
relations  which  exist  between  the  yeasts  and  Endomycetes,  Van 
Tieghem,1  in  his  recent  classification,  has  attempted  to  place  them 
in  one  group,  the  Eremascines.  Hansen,  on  the  contrary,  considers 
the  yeasts  as  making  up  a  special  family  of  Ascomycetes  related  to 
Exoascus  and  Endomycetes  which  he  calls  Saccharomycetes.  Although 
among  the  Endomycetes  and  Saccharomycetes  there  exist  all  degrees 
of  transition,  and  although  there  are  such  varieties  as  End,  javanensis, 
which  is  with  difficulty  attached  to  either  groups  of  these  two  families, 
we  believe  with  Schroter2  that  the  yeasts  should  be  made  a  distinct 
family  closely  related  to  the  Endomycetes  and  making  up  the  group 
Protoascines.  The  great  number  of  the  yeasts  and  their  medical  and 
industrial  significance  seem  to  justify  this  opinion.  How  shall  the 
representatives  of  the  Saccharomycetes  be  grouped?  Profiting  by  the 
morphological  investigations  of  these  later  years  by  Hansen,3  a  clas- 
sification may  be  proposed.  This  classification  which  was  proposed 
in  1904  and  has  been  added  to  by  the  work  of  Klocker  4  and  Lindner,5 
is  today  uniformly  accepted. 

However  the  recent  work  on  the  systematic  and  phylogenic  rela- 
tions of  the  yeasts,  not  considering  the  great  lines  of  this  classifica- 
tion, do  not  justify  it  completely.  We  shall  adopt  a  classification  a 
little  different  from  that  of  Hansen's. 

1  Van  Tieghem.    Elements  de  botanique.    Masson  et  Cie.,  Paris,  4th  Edition, 
1906. 

2  Schroter,  J.  in  Engler  and  PrantFs  Die  nat.  Pflanzen.   W.  Englemann,  Leipzig, 
1889. 

3  Hansen,  E.  C.     Grundlinien  und  Systematik  der  Saccharomyceten.     Cent. 
Bakt.  12  (1904),  528. 

4  Klocker,   A.     Deux  nouveaux  genres  de  la  famille   des  Saccharomycetes. 
Comp.  Rend,  des  trav.  du  lab.  de  Carlsberg,  8,  Book  4,  1909. 

5  Lindner,  P.    Mikroskopische  Betriebskontrolle  in  den  Garungswerben.     Paul 
Parey,  Edit.  Berlin,  6th  edition,  1909. 

189 


190  CLASSIFICATION   OF  THE  YEASTS 

We  shall  eliminate  from  the  Saccharomycetes  all  of  the  yeasts  which 
do  not  form  ascospores.  Such  are  the  Torula,  Mycoderma,  and  the 
pathogenic  yeasts.  These  yeasts  do  not  offer  any  characteristic  which 
permits  giving  them  an  accurate  place  in  classifications  of  fungi. 
The  one  may  represent  forms  derived  from  mycelial  fungi  and  fixed 
in  the  state  of  yeasts,  the  others  may  be  true  yeasts  which  have 
become  asporogenic.  They  may  be  placed  apart  in  a  separate  group 
from  the  Saccharomyces. 

In  the  family  of  Saccharomyces,  we  shall  include  all  yeasts  which 
sporulate  whatever  their  mode  of  division.  Contrary  to  Hansen, 
we  shall  not  separate  the  Schizosaccharomyces.  These  yeasts,  if  they 
are  differentiate  from  the  other  yeasts  by  the  mode  of  division 
(transverse  partition),  belong  incontestably  to  the  Saccharomycetes 
by  the  copulation  which  preceded  the  formation  of  the  asc  with  the 
greater  part  of  them.  They  are  related  to  the  Saccharomycodes  in 
which  the  cells  divide  by  an  intermediate  method  between  typical 
partition  and  budding  and  which  offer  a  form  of  transition  between 
the  Schizosaccharomyces  and  other  yeasts.  We  shall  subdivide  the 
Saccharomyces  into  five  groups. 

The  first  group  will  include  the  Schizosaccharomyces  characterized 
by  their  method  of  division,  transverse  partition.  By  the  formation 
of  the  asc  which  results  from  an  isogamic  copulation,  this  group 
may  be  regarded  as  strictly  related  to  the  Endomycetes. 

In  the  second  group  are  placed  those  yeasts  which  offer  in  the  origin 
of  the  ascs,  a  copulation  iso-  or  heterogamic  and  which,  having  lost  their 
sexuality,  have,  however,  preserved  traces  of  it.  It  is  a  very  primitive 
group  from  which  seem  to  be  derived  all  other  budding  yeasts. 

In  the  third  group,  we  find  all  yeasts  in  which  the*  formation  of 
the  asc  is  not  preceded  by  any  sexual  phenomenon  and  which  in  liquid 
media,  vegetate,  at  first,  as  a  sediment  and  produce  later  a  scum 
very  slowly  more  or  less  mucous.  In  certain  species,  a  parthenogamy 
between  ascospores  may  intervene.  Almost  all  of  the  species  in  this 
group  are  able  to  induce  fermentations.  This  group  corresponds  to 
Hansen's  first  group  less  the  yeasts  of  our  second  group. 

In  the  fourth  group  are  yeasts  which,  without  any  trace  of  sex- 
uality in  the  formation  of  the  asc,  form  in  liquid  carbohydrate  media 
a  mycodermic  scum.  After  the  air  has  penetrated  into  its  interstices, 
it  takes  on  a  dry  opaque  appearance.  Most  of  these  yeasts  do  not 
cause  fermentations  but  produce  ethers.  Some  of  them  have  parthe- 
nogamy between  the  ascospores.  This  group  corresponds  to  Hansen's 
second  group.1 

1  The  classification  of  Hansen  differs  from  ours  only  in  the  following  points: 
First,  the  Schizosaccharomyces  are  excluded  and  considered  as  a  special  group  of 


CLASSIFICATION  OF  THE  YEASTS  191 

Finally  in  the  fifth  group  (Saccharomycetes  doubted  by  Hansen) 
we  shall  include  the  genera  Monospora,  Nematospora,  and  Coccidiascus, 
which  offer  by  the  shapes  of  their  ascospores  very  special  charac- 
teristics and  of  which  the  affinities  are  not  well  known. 

The  first  group  includes  only  the  genus  Schizosaccharomyces  repre- 
sented by  only  a  few  species. 

In  the  second  group  we  shall  place  the  genus  Zygosaccharomyces, 
first  characterized  only  by  isogamic  or  heterogamic  copulation  which 
precedes  the  formation  of  the  ascs  and  which  seems  to  make  up  with 
the  Schizosaccharomyces  an  archaic  type  which  has  retained  an  an- 
cestral copulation  similar  to  the  Eremascus.  Next  comes  the  genus 
Debaromyces  (Klocker)  characterized  by  its  ascopores  in  thorny  mem- 
brane. This  genus  actually  includes  only  a  single  species  Deb.  glo- 
bosus  which  has  a  copulation  similar  to  the  Zygosaccharomyces  and 
appears  to  progress  toward  heterogamy.  The  new  genus  Nadsonia 
(Guilliermondia)  of  Nadson  and  Kinokotine,  created  for  species  with 
heterogamic  copulation,  is  characterized  by  the  fact  that  the  asc  is  formed 
from  a  cell  and  a  bud  from  that  same  cell.  The  ascospores,  generally 
to  the  number  of  one,  resemble  the  ascospores  of  Debaromyces.  They 
have  a  large  fat  globule  in  their  center  and  a  membrane  slightly 
verrucose.  The  genus  Schwanniomyces  (Klocker)  includes  only  Sch. 
ocddentalis  which  is  characterized  by  a  thorny  membrane  but  belted 
by  a  projecting  collar.  Here  the  ascs  have  preserved  traces  of  sexual 
attraction  and  attempt  to  anastomose  two  by  two  before  sporulation. 
The  genus  Torulaspora,  created  recently  by  Lindner  for  yeasts  which 
present  the  typical  spherical  shape  of  the  Torula,  is  badly  defined; 
however,  we  shall  reserve  for  it  a  place  along  with  the  Schwanniomyces 
because  the  •  ascogenic  cells  show  traces  of  sexual  attraction.  We 
shall  include  in  this  genus,  by  the  side  of  Torulaspora  Delbriicki 
(Lindner),  a  certain  number  of  yeasts  which  offer  equally  a  spherical 
shape  and  which,  on  the  other  hand,  have  preserved  traces  of  sexual 
attraction  (yeasts  E  and  F  of  Rosa  for  instance) . 

The  third  group,  one  large  in  numbers,  includes  the  genus  Sac- 
charomycodes  (Hansen)  in  which  the  cells  multiply  by  a  process  in- 
termediary between  transverse  partition  and  budding,  and  which, 
from  this  point  of  view,  may  be  regarded  as  a  form  of  transition 


yeasts.  Secondly,  the  Saccharomyces  are  divided  into  two  groups  only;  the  first 
includes  yeasts  which  form  a  scum  only  at  the  end  of  fermentation.  This  scum 
is  mucous  without  occluded  air  bubbles.  This  group  includes  the  genera  Sac- 
charomyces, Zygosaccharomyces,  Saccharomycodes,  Saccharomycopsis.  The  second 
group  includes  the  types  which  give  a  scum  at  the  beginning  with  bubbles  of  air 
in  it:  genera,  Willia  and  Pichia.  Thirdly,  the  genera  Nematospora  and  Monospora 
make  up  a  group  under  the  name  of  doubtful  Saccharomycetes. 


192  CLASSIFICATION   OF  THE  YEASTS 

between  the  Schizosaccharomyces  and  the  ordinary  yeasts.  This  genus 
is  characterized  by  a  tendency  to  produce  mycelial  formations  rather 
well  developed  and  the  replacement  of  ancestral  sexuality  by  a  com- 
pensating phenomenon  or  parthenogamy  consisting  in  the  fusion  of 
ascospores  two  by  two. 

After  this  genus  may  fall  the  genus  Saccharomycopsis  (Schion- 
ning)  which  only  includes  S.  guttulatus  and  which  is  characterized 
by  ascospores  in  a  double  membrane  resembling  those  of  the  Endo- 
mycetes  (Eremascus,  End.  fibuliger,  and  capsularis) .  We  shall  separate 
the  Saccharomycopsis  capsularis  (Schionning)  from  this  genus  in  order 
to  include  it  with  the  genus  Endomyces.  '  The  investigations  of  Guil- 
liermond  with  this  species  seem  to  indicate  that  it  approaches  E. 
fibuliger  when  the  mycelial  formation  and  method  of  formation  of  the 
ascs  are  considered. 

The  genus  Saccharomyces  (Meyen)  includes  all  yeasts  in  which  the 
formation  of  a  mycelium  is  not  observed  and  in  which  sexuality  has 
disappeared  with  the  exception  of  a  few  species  (yeast  Johannisberg 
II)  in  which  the  primitive  copulation  has  been  replaced  by  a  fusion 
between  the  ascospores  (parthenogamy). 

Finally  the  genus  Hansenia  (Lindner-Klocker)  characterized  by 
its  special  apiculate  cells  and  hat-shaped  ascospores  terminates  the 
series. 

Since  the  Saccharomyces  includes  all  brewery,  distillery,  cider 
and  wine  yeasts  and  other  industrial  yeasts,  and  consequently  a 
large  number,  we  shall,  with  Hansen,  make  six  sub-groups  according 
to  their  fermentation  reactions:  First,  Saccharomyces  which  ferment 
saccharose,  maltose  and  dextrose  with  no  action  on  lactose.  Sec- 
ondly, Saccharomyces  which  ferment  saccharose  and  dextrose  but  do 
not  ferment  lactose  or  maltose.  Thirdly,  Saccharomyces  which  fer- 
ment dextrose  and  maltose  but  not  saccharose  and  lactose.  Fourthly, 
Saccharomyces  which  ferment  dextrose  but  neither  lactose,  saccharose, 
or  maltose.  Fifthly,  Saccharomyces  which  ferment  lactose.  Sixthly, 
Saccharomyces  which  produce  no  fermentation  and  in  which  the  fer- 
menting function  is  imperfectly  known. 

In  the  fourth  group,  we  shall  include,  with  Hansen,  two  genera, 
Pichia  (Hansen),  characterized  by  hemispherical  ascospores,  and 
Willia,  characterized  by  special-shaped  ascospores  having  the  form 
of  a  derby  hat. 

In  the  fifth  group,  we  shall  place  the  genus  Monospora  (Metsch- 
nikoff),  characterized  by  its  ascs  with  a  single  ascospore,  and  the  genus 
Nematospora  (Peglion),  characterized  by  its  asc  with  8  fusiform  asco- 
spores with  a  long  mycelium  and  the  genus  Coccidiascus  (Leger), 
which  is  characterized  by  a  probable  copulation  preceding  the  forma- 


CLASSIFICATION   OF  THE  YEASTS  193 

tion  of  the  asc   containing  8  ascospores.     These  two  genera,   until 
their  relations  are  better  known,  merit  a  place  apart. 

Along  with  the  Saccharomycetes  we  shall  make  up  a  family  of  non- 
Saccharomycetes  or  doubtful  yeasts  —  all  those  which  do  not  form 
spores.  Three  groups  will  be  made  here:  First,  the  Torula,  including 
all  yeasts  which  in  liquid  media  vegetate  in  the  bottom  of  the  culture 
tube  but  eventually  form  a  slimy  scum  with  no  air  bubbles,  having 
all  of  the  other  characteristics  of  the  third  group  with  the  exception 
of  spore  formation.  Secondly,  the  genus  Pseudosaccharomyces  pro- 
posed by  Klocker  for  the  apiculate  yeasts  which  do  not  sporulate 
and  the  Mycoderma  which  forms  a  slimy  scum  with  air  bubbles.  These 
correspond,  in  general,  with  the  fourth  group  of  the  Saccharomycetes. 
Thirdly,  the  genus  Medusomyces  (Lindau),  characterized  by  a  thick, 
stratified,  gelatinous  scum,  and  the  pathenogenic  yeasts  to  which  have 
been  given  the  generic  name  of  Cryptococcus  (Vuillemin).1  Below 
is  given  a  resume*  of  the  classification  which  we  have  just  outlined. 

Family   of  Saccharomycetes 

Unicellular  fungi,  multiplying  by  budding,  sometimes  by  parti- 
tion and  forming  ascs.  Each  cell  may  change  into  an  asc  in  which  are 
formed  from  1  to  4,  rarely  12,  ascospores,  each  ascospore  enclosed  in 
a  vegetative  cell. 

FIRST  GROUP 

Yeasts  multiplying  by  partition.  Ascs  often  derived  from  a  copu- 
lation, with  4  or  8  ascospores.  These  are  provided  with  a  single  mem- 
brane. 

Genus  I.   Schizosaccharomyces 

SECOND   GROUP 

Budding  yeasts;  sexual  phenomena,  sometimes  only  in  traces,  in 
the  formation  of  the  asc. 

1  De  Beurmann  and  Gougerot  (Les  mycoses  dans  le  nouveau  Traite  de  mede- 
cine  et  de  therapeutique  de  A.  Gilbert  et  Thoinot,  Bailliere,  ed.  Paris,  1910) 
have  created  the  following  three  genera  for  pathogenic  yeasts  which  do  not  sporu- 
late. 

1.  Atelossaccharomyces  (areXos  =  imperfect)  which  include  all  well-differenti- 
ated yeasts  which  do  not  sporulate. 

2.  Parasaccharomyces  which  include  fungi  resembling  the  yeasts  but  which 
offer  rudimentary  filamentous  forms  sometimes  true  filaments. 

3.  Zymonema  (£vfj.rj   =  levure,  VYUJLCL   =  filament)   which  include   intermediate 
forms  between  the  yeasts  and  Endomycetes  characterized  by  a  mixture  of  yeast 
forms  and  mycelial  formations. 

The  pathogenic  yeasts  are,  as  stated  above,  not  very  well  known  and  the 
placing  of  them  into  genera  is  difficult.  This  classification  seems  premature. 


194  CLASSIFICATION.  OF  THE  YEASTS 

Genus  II.   Zygosaccharomyces.   Barker 

Ascs  preceded  by  a  copulation,  iso-  or  heterogamic,  ascospores 
with  a  thick  membrane. 

Genus  III.    Debaromyces.   Klocker 

Ascs  derived  from  a  copulation  most  often  heterogamic,  with 
globular  ascospores  provided  with  a  single  verrucose  membrane. 

Genus  IV.   Nadsonia.    (Guilliermondia)  Nadson 

Ascs  derived  by  budding  from  a  cell  formed  by  heterogamic  copu- 
lation. Ascs  with  walls  more  or  less  thick. 

Genus  V.   Schwanniomyces.   Klocker 

Traces  of  copulation ;  ascospores  with  a  single  verrucose  mem- 
brane formed  of  two  unequal  parts  girdled  and  provided  with  a  pro- 
jecting collar. 

Genus  VI.   Torulaspora.   Lindner  1 

Round  cells  resembling  Torula  with  a  large  fat  globule  in  the  center. 
The  ascs  present  only  traces  of  copulation  in  their  origin. 

THIRD  GROUP 

Budding  yeasts  which  form,  in  sugar  solutions,  at  first  a  deposit, 
and  later  on  a  more  or  less  slimy  scum  without  occluded  air.  Asco- 
spores, round  or  oval,  with  from  1  to  2  membranes,  germinating  by 
budding;  generally  produce  alcohol. 

Genus  VII.   Saccharomy codes.   Hansen 

Cells  divide  by  a  procedure  intermediary  between  budding  and 
division.  Frequently  rudiments  of  a  mycelium  with  transverse  walls. 

Ascospores  in  a  single  membrane  germinating  in  a  single  direction 
in  the  form  of  a  tube  which  swells  up  and  separates  the  ascospore  by 
the  formation  of  a  transverse  wall  accompanied  by  a  slight  circular 
constriction.  Germination  often  preceded  by  parthenogamy. 

Genus  VIII.  Saccharomy copsis.  Klocker 
Ascospores  in  two  membranes. 

1  This  genus  seems  to  be  characterized  by  its  traces  of  copulation  as  the 
investigations  of  Rose  have  indicated.  We  have  united  these  characteristics  with 
those  provided  by  Linder  to  characterize  this  geniis. 


CLASSIFICATION   OF  THE  YEASTS  195 

Genus  IX.     Saccharomyces.     Meyen 

Ascospores  in  a  single  membrane,  germinating  by  budding  some- 
times with  the  formation  of  a  rudimentary  mycelium. 

First  Sub-Group :  Saccharomyces  fermenting  dextrose,  maltose 
and  saccharose,  but  not  lactose. 

Second  Sub-Group :  Saccharomyces  fermenting  dextrose  and  sac- 
charose but  neither  maltose  nor  lactose. 

Third  Sub-Group:  Saccharomyces  fermenting  dextrose  and  mal- 
tose but  neither  saccharose  nor  lactose. 

Fourth  Sub-Group :  Saccharomyces  fermenting  dextrose  but  not 
maltose,  saccharose  nor  lactose. 

Fifth  Sub-Group:    Saccharomyces  fermenting  lactose. 

Sixth  Sub-Group:  Saccharomyces  not  inducing  fermentations  or  in 
which  the  characteristics  of  fermentations  are  insufficiently  known. 

Genus  X.     Hansenia.     Lindner.     Klocker 
Apiculate  cells.     Ascs  with  a  single  ascospore. 

FOURTH  GROUP 

Budding  yeasts  which  form  a  scum  immediately  in  sugar  media; 
scum  is  dry  and  opaque,  including  air.  Ascospores  in  characteristic 
shapes  (in  the  form  of  a  lemon,  hat  or  angulous)  with  a  single  mem- 
brane, often  with  a  projecting  collar.  Generally  do  not  produce 
alcohol  but  ether. 

Genus  XI.     Pichia.     Hansen 

Hemispherical  ascospores.  Rudimentary  mycelium  well  developed. 
Do  not  cause  fermentations. 

Genus  XII.     Willia.     Hansen 

Ascospores  in  the  form  of  a  lemon  or  hat  with  a  projection  like 
a  girdle;  generally  do  not  produce  alcohol  but  ether. 


FIFTH  GROUP 
Yeasts  in  which  the  relationships  are  not  well  known. 

Genus  XIII.     Monospora.     Metschnikoff 

Budding  yeasts,  ascs  with  a  single  ascospore,  in  the  form  of 
needle,  germinating  laterally  by  budding. 


196  CLASSIFICATION   OF  THE  YEASTS 

Genus  XIV.     Nematospora.     Peglion 

Budding  yeasts,  ascs  with  4  fusiform  ascospores,  terminating  with 
a  cilium. 

Family  of  Non-Saccharomycetes 
Budding  yeasts  but  forming  no  ascs. 

Genus  I.     Torula.     Turpin 

Generally  spherical  cells,  often  forming  a  scum  but  only  after 
fermentation;  scums  always  slimy  without  the  presence  of  air  bubbles. 

Genus  II.     Pseudosaccharomyces.     Klocker 
Apiculate  cells. 

Genus  III.    Mycoderma.     Persoon 

Cells  generally  elongated;  scums  are  formed  at  the  end  of  devel- 
opment with  the  presence  of  air  bubbles. 

Genus  IV.    Medusomyces.     Lindau 
Scums  appearing  at  the  beginning  thick,  stratified  and  gelatinous. 

Genus  V.     Cryptococcus.     Kutzing-Vuillemin 
Yeasts  without  ascs,  parasitic  to  animals. 

There  now  remains  a  descriptive  study  of  species  of  yeasts.  We 
shall  describe  all  of  the  yeasts  which  are  actually  known.  The  yeasts 
are  excessively  numerous  and  space  will  not  permit  an  examination  of 
all  of  them.  Many  of  the  industrial  yeasts,  well  known  on  account  of 
their  physiological  properties,  have  not  been  studied  morphologically 
and  have  not  been  given  provisional  names.  Finally  many  of  the 
Torula  and  Mycoderma  and  pathogenic  yeasts  have  been  insuffi- 
ciently characterized.  It  will  be  necessary  to  pass  over  those  which 
are  not  completely  described  and  devote  our  attention  to  those  which 
are  more  fully  characterized. 


PART  II  — STUDY  OF  SPECIES 

CHAPTER  X 
FAMILY  OF  SACCHAROMYCETACEAE 

UNICELLULAR  fungi,   multiplying   by  budding  or  transverse 
division  and  forming  ascs.    Each  cell  has  the  ability  to  change 
into  an  asc  and  form  from  one  up  to  twelve  ascospores,  each 
ascospore  germinating  and  forming  a  vegetative  cell. 

FIRST  GROUP 
Genus  I.    Schizosaccharomyces 

Round  or  rectangular  cells,  dividing  by  transverse  partition.  Asc 
with  4  or  8  ascospores  ordinarily  resulting  from  isogamic  copula- 
tion. 

SCHIZOSACCHAROMYCES  OCTOSPORUS.    Beijerinck l 

This  species  was  found  by  Beijerinck  on  fruits  from  warm  climates 
(raisins  from  Corinth,  Greece,  Asia  Minor,  and  Turkey,  and  figs  from 
Smyrna).  It  possesses  large  cells  of  various  shapes;  some  are  rectan- 
gular, resembling  the  oidia  of  Endomyces  or  giant  bacteria;  others  are 
spherical  and  resemble  the  Micrococcus  (Figs.  8  and  14).  The  rec- 
tangular cells  predominate  in  young  cultures,  while  the  spherical  cells 
appear  especially  when  multiplication  commences,  and  change  into 
an  asporogenic  type. 

Often  the  cells  near  the  ends  show  the  presence  of  circular  lines 
which  mark  the  divisions  between  the  old  part  of  the  cell  wall  and  that 
which  was  newly  formed. 

Multiplication  is  brought  about  by  transverse  division:  a  wall 
appears  in  the  middle  of  the  cell,  making  two  daughter  cells.  The 
wall  quickly  increases  in  size  and  the  two  cells  become  round  in  shape. 
The  cells  may  remain  attached  in  such  a  way  that  the  mother  cell 
may  have  4  or  more  daughter  cells  attached.  The  daughter  cells 
may  undergo  a  transverse  partition  without  separating  from  the 
mother  cell.  The  cells  are  then  grouped  somewhat  in  the  same  way 
as  the  Sartina. 

Sch.   octosporus  never  contains  glycogen  at  any  time  of  its  de- 

1  Beijerinck,  W.     Sch.  octosporus.    Cent.  Bakt.  16,  1896,  also  1897. 

197 


198  FAMILY  OF  SACCHAROMYCETACEAE 

velopment.  Speculation  seems  to  be  rapid  and  appears  at  the  end  of 
two  or  three  days  on  solid  media  (slices  of  carrot,  beer  wort  or  wine 
to  which  gelatin  has  been  added).  It  may  happen  that,  at  the  end 
of  fermentation,  the  vegetative  cells  in  the  sediment  may  contain 
ascospores  but,  in  this  case,  sporulation  is  feeble.  The  ascs  form 
with  difficulty  on  plaster  of  Paris  blocks;  according  to  Seiter  they 
appear  at  the  end  of  six  or  seven  hours  at  25°  C. 

Sporulation  is  preceded  by  a  sexual  phenomenon  which  was 
studied  by  Guilliermond  l  in  1901.  The  asc  results  from  an  isogamic 
copulation  which  takes  place  between  two  neighboring  cells.  These 
unite  by  means  of  a  copulation  canal  through  which  the  contents  of 

the  two  cells  mix.  The  fusion  results  in  the 
formation  of  a  large  oval  zygospore  (6-10.5 
wide  and  14-20.5  long).  This  transforms 
slowly  into  an  asc.  Sometimes  the  fusion 
remains  incomplete,  and  the  asc  seems  to  be 
formed  of  two  enlarged  parts  united  by  a 
canal.  All  intermediary  stages  are  found, 
however,  between  complete  and  incomplete 
fusion  (Figs.  14,  15,  and  16). 

The  ascospores,   always  4  or  8  in  each 
asc,  are  usually  ellipsoidal  in  shape.     They 
FigTT^T-Karyokenesism   are    surrounded    by   a   membrane,    covered 
the  Ascs  of  Schizosac-  with  a  starchy  reserve  material  which  stains 
charomycesOctosporus.          blue  with  io(Jine  (Lindner);  this  is  utilized 

during  germination.  The  wall  of  the  asc  persists,  or  more  often  disap- 
pears immediately,  before  germination;  the  ascospores,  having  been  set 
free,  separate  or  remain  attached.  Germination  begins  by  a.  swelling 
of  the  ascospores  which  take  the  appearance  of  vegetative  cells  and 
divide  in  the  usual  manner.  On  nutrient  gelatin  the  colonies  are  round 
with  a  thick  center. 

We  have  seen  that  Beijerinck  noticed,  when  the  yeast  was  inoc- 
ulated onto  gelatin,  that  three  sorts  of  colonies  were  obtainable;  first, 
white  colonies  made  up  of  cells  which  formed  ascospores;  secondly, 
clear  brown  colonies  made  up  of  only  vegetative  cells;  thirdly,  light 
brown  colonies  made  up  of  cells  forming  ascs  and  those  with  no  ascs. 

1  Guilliermond,  A.  Recherches  histologique  sur  la  sporulation  des  Schizo- 
saccharomycetes.  Comp.  Rend.  Acad.  Sciences,  133,  1901 ;  Recherches  cytologiques 
sur  les  levures  et  quelques  moisissures  a  form  levures.  These  for  the  Doctorate 
in  Sciences  at  the  Sorbonne,  Storck,  Lyon,  1902.  Summarized  in  the  Revue 
generale  de  Botanique,  15,  1903;  Recherches  sur  le  developpement  du  Gloeosporium 
nervisequum  et  sa  pretendue  transformation  en  levures.  Rev.  gen.  de  Bot.  20, 
1909. 


SCHIZOSACCHAROMYCES  POMBE  199 

This  indicated  the  possibility  of  two  types  of  yeasts,  a  sporogeriic  and 
a  non-sporogenic  race.  The  light  brown  colonies  are  formed  by  a 
mixture  of  the  two  types. 

Both  types  are  encountered  constantly  in  nature.  They  possess 
different  morphology  and  physiology.  The  sporogenic  race  is  made 
up  of  rectangular  cells  while,  on  the  contrary,  in  the  asporogenic  type, 
round  cells  predominate  and  are  often  situated  in  the  shape  of  a 
Sarcina.  Finally  the  sporogenic  race  liquefies  gelatin  more  rapidly 
than  the  asporogenic.  The  sporogenic  race  shows  a  tendency  to  trans- 
form into  the  asporogenic  type  when  cultivated  for  a  long  time  in 
the  laboratory. 

Schizosaccharomyces  octosporus  never  produces  a  scum  on  beer  wort 
but  simply  a  feeble  ring.  It  ferments  lactose,  dextrose,  levulose, 
d-galactose,  d-mannose,  raffinose,  dextrine  and  a-methylglucoside.  It 
may  also  cause  a  feeble  fermentation  of  xylose  (Lindner).  It  has  no 
action  on  saccharose  which  it  does  not  ferment.  From  the  biochemical 
point  of  view,  Schizosaccharomyces  octosporus  is  distinguished  from 
Saccharomyces  mellacei  and  Sch.  Pombe  in  that  it  ferments  d-galac- 
tose and  has  no  action  on  saccharose  nor  inuline. 

SCHIZOSACCHAROMYCES  POMBE.    Lindner 

Schizosaccharomyces  Pombe  was  discovered  by  Saare  and  Zeidler  in 
African  beer  made  from  millet  and  described  by  Lindner  l  in  1893. 
Its  cells  are  much  smaller  than  those  of  Schizosaccharomyces  octo- 
sporus, usually  retangular,  with  rounded  ends  and  about  7  /LI  in  length 
and  4.5  /z  broad.  The  cells  resemble  the  Oidia  of  the 
Endomyces  very  much  or  even  giant  bacilli.  In  old 
media  they  tend  to  decrease  in  length  and  approach  the 
appearance  of  bacteria. 

They  divide  by  transverse  partition  always  in  the 
same  way  as  Schizosaccharomyces  octosporus  (Fig.  75). 
The  transverse  walls  separate  the  cells  into  unequal  Fig.  75.  —  Vege- 


parts.    Under  certain  conditions,  as  the  absence  of  air,      ^JX6  Cells  in 
f  Schizosaccha- 

tne  cells  elongate  very  much  and  may  present  many      romyces  Pombe 

cross  walls  without  any  separation  taking  place.  Some-  on  Carrot 
times  one  may  observe  the  formation  of  lateral  branches. 
We  have  seen  that  Lepeschkin  has  been  able  to  obtain  on  beer  wort, 
in  the  deposit,  little  floes  having  a  characteristic  mycelial  formation 
with  cross  walls  and  branchings.  The  cells  of  this  yeast  never  include 
glycogen.  Growth  demands  at  least  a  temperature  of  15°  C. 

1  Lindner,   P.     Schizosaccharomyces  Pombe  no.  sp.  ein  neucr  Garungserreger 
Wochenschr.  Brauerei.     1893. 


200  FAMILY  OF  SACCHAROMYCETACEAE 

Sporulation  is  accomplished  with  difficulty  on  plaster  blocks,  but 
is  easily  observed  in  hanging  drops  in  which  it  appears  at  the  end  of 
seven  or  nine  hours.  It  is  easily  obtained  on  slices  of  carrot  at  the 
end  of  a  few  hours,  in  old  cultures  on  gelatin,  and  in  the  growth  in 
beer  wort  after  fermentation.  Guilliermond  l  has  found  that  the  ascs 
result  from  an  isogarnic  copulation.  The  fusion  is  always  incomplete 
and  results  in  the  formation  of  two  swelled  parts  united  by  a  narrow 
canal.  The  ascospores,  always  to  the  number  of  four,  are  formed  in 
pairs,  two  in  each  swelling.  Their  dimensions  are  about  4  JJL  in  diame- 
ter (Fig.  76).  Their  walls  are  covered  with  a  starchy  substance  which 

is  colored  blue  with  iodine. 
Quite  often  the  ascospores  may 
form  at  the  expense  of  cells  which 
have  not  undergone  copulation. 
The  ascs  reabsorb  their  mem- 
branes generally  before  germina- 
tion, and  thus  free  the  ascospores. 
Germination  is  brought  about  in 
the  following  manner:  the 
ascospores  swell  up  and  divide 
by  transverse  partition  like  the 
vegetative  cells. 

Beijerinck  has  noticed  as  in  Sch.  octosporus,  the  existence  of  a 
sporogenic  variety  forming  white  colonies  on  gelatin  and  an  asporo- 
genic  variety  forming  brown  colonies  on  the  same  medium. 

This  yeast  forms  no  scum  on  beer  wort  but  produces  a  ring  at 
the  end  of  a  month.  On  gelatin,  there  develops  a  compact  layer  of 
fine  channelings  with  a  liquefaction  of  this  medium. 

Sch.  Pombe  is  a  yeast  easily  attenuated.  Fermentation  is  very  active 
and  manifests  itself  as  top  fermentation.  The  optimum  temperature  for 
fermentation  is  situated  between  30°  and  35°  C.  This  yeast  provokes 
an  apparently  strong  fermentation  of  beer  wort:  it  ferments  maltose, 
saccharose,  dextrose,  levulose,  raffinose,  and  a-methylglucoside.  On 
the  other  hand,  it  is  able  to  ferment  inuline  and  dextrine. 

SCHIZOSACCHAROMYCES  MELLACEI.  Jorgensen2 

This  yeast  was  discovered  by  Greg  3  from  Jamaican  molasses  used 
in  the  manufacture  of  rum.  It  has  been  described  by  Jorgensen  and 
Holm.  It  is  a  species  closely  related  to  Schizosaccharomyces  Pombe 

1  See  references  under  this  subject  for  Sch.  octosporus. 

2  Jorgensen,  A.     Die  Mikroorganismen  der  Garungsindustrie,  5th  edition  P. 
Parey,  Berlin,  1909. 

3  Greg,    P.    The   Jamaica   Yeasts.     Bulletin   of   the    Botanical  Department, 
Jamaica,  Vol.  2,  1895. 


SCHIZOSACCHAROMYCES   MELLACEI 


201 


tative  Cells  of 
Schizosaccha- 
romyces  Mel- 
lacei from 
Cultures  on 
Slices  of  Car- 
rot. 


from  which  it  may  scarcely  be  distinguished  by  its  morphological 
characteristics.  The  cells  have  the  same  shape  as  those  of  Sch. 
Pombe  but  are  generally  a  little  larger  (Fig.  78). 

Developing   on   carrot    slants,   Guilliermond   found   the   measure- 
ments to  be  about  9.5  jit  by  5.1  ju;   the  cells  of  Sch. 
Pombe  under  the  same  conditions  are  about  7  ju  long      /  /  \  i    p, 
and  4.5  M  wide  (Fig.  77).  l/\\V0 

Lepeschkin  has  noticed  in   this   species   as   in   the 
former   one,    the   appearance   of   a   true   mycelium   in      ~  ,  , 
deposits  at  the  bottom  of  the  culture  flask.  M    ^< 

Sporulation  of  this  yeast  is  obtained  easily  at  the  Fig  77 Vege- 

end  of   a   few  days  on  beer  wort  gelatin  in  hanging 
drops.     The  observations  of  Guilliermond1  have  shown 
that  the  ascs  result  from  an  isogamic  coplation  which  is 
accomplished  in  the  same  manner  as  with  Sch.  Pombe. 
The  ascs  have  a  shape  like  a  dumb  bell,  both  swelled 
portions  being  connected  by  a  narrow  canal  (Fig.  78). 
The  ascospores  always  to  the  number  of  four  appear  in  pairs  in  each 
large  part  of  the  asc.     Cases  of  parthenogenesis  may  be  often  observed 
in  which  the  ascs  result  without  previous  copulation.     The  ascospores 
are  long  and  rounded  (about  4/4  in  diameter).     They  are  very  refractive, 

and  are  clothed  with  a  mem- 
brane impregnated  with  starchy 
materials  which  are  stained  blue 
CV  by  iodin  in  potassium  iodide. 
U  This  yeast  produces  no  scum 
on  beer  wort  but  forms  a  ring 
at  the  end  of  four  or  five  months. 
Plate  cultures  in  gelatin  and 
streaks  on  gelatin  offer  growths 
which  on  the  surface  and  in  the 
medium  are  closely  detailed. 
Sch.  mellacei  ferments  beer  wort 
at  25°;  there  are  signs  of  top 

fermentation  with  a  broken-up  deposit,  not  very  compact.  During 
fermentation  it  liberates  an  agreeable  odor.  It  ferments  dextrose, 
maltose,  levulose,  saccharose,  raffinose,  d-mannose,  dextrine,  a-methyl- 
glucosides  and  inulin;  it  is  distinguished  from  Sch.  Pombe  by  the  fact 
that  it  ferments  d-mannose  on  which  the  latter  has  no  action. 

According  to  Greg  this   yeast  produces  several  types  which  are 
characterized  by  the  peculiar  odor  given  off  during  fermentation,  or 

1  Guilliermond,   A.     Rem.  sur  la  copulation  du  Schizosaccharomyces  mellacei. 
Bull.  Soc.  Bot.  de  Lyon,  1903. 


CD 

Fig.  78.  —  Formation  of  the  Asc  in  Schizosac- 
charomyces Mellacei. 


202  FAMILY  OF  SACCHAROMYCETACEAE 

by  the  amount  of  alcohol,  which  varies  between  6.6  and  7.6  per  cent  by 
volume.  These  types  also  differ  by  the  rate  at  which  cellular  multi- 
plication takes  place. 

It  seems  appropriate  to  mention  a  very  interesting  Schizosaccharo- 
myces  which  we  l  have  been  able  to  observe.  This  was  sent  by  Pro- 
fessor Beijerinck  under  the  name  of  Sch.  mellacei.  An  examination 
of  this  yeast  shows  that  it  differs  from  Sch.  mellacei  by  a  complete 
disappearance  of  sexual  processes.  (Fig.  79.)  The  ascospores,  al- 
ways to  the  number  of  four,  are  formed  in 
ordinary  rectangular  or  elongated  cells  with- 
out any  previous  copulation.  The  vegeta- 
tive cells  are  much  smaller  than  those  of 
Sch.  mellacei  or  Sch.  Pombe.  On  carrot  slants 
their  average  size  is  6.8  ju  long  and  3.5  ju  wide. 
The  ascospores  are  about  the  same  size  (about 

Fig.     79.  —  Parthenogenetic   4  jit)  as    those    of     Sch.    mellacei     and    Sch. 
Variety  of  Schizosaccharo-    pombe 
myces  Mellacei. 

By  its  morphological  characters,  it  differs 

from  Sch.  mellacei  and  Sch.  Pombe.  Unfortunately  no  study  was  un- 
dertaken of  its  biochemical  features,  and  we  are  not  able  to  state 
whether  it  is  a  new  species  or  whether  it  is  a  variety  of  Sch.  mellacei 
and  Sch.  Pombe  in  which  sexuality  has  disappeared. 

SCHIZOSACCHAROMYCES  ASPORUS.   Eykmann 

This  yeast  has  been  described  by  Eykmann; 2  it  is  a  yeast  used  in 
the  manufacture  of  arrack  (the  alcoholic  drink  of  Java  made-  from  mo- 
lasses from  sugar  refineries  and  rice  powder).  It  is  distinguished  from 
Schizosaccharomyces  Pombe  by  the  fact  that  it  does  not  produce  endo- 
spores.  Beijerinck  thinks  that  it  is  an  asporogenic  variety  of  Sch. 
Pombe.  On  nutrient  gelatin,  it  produces  white  and  brown  colonies; 
the  white  colonies  give  more  ascospores  than  the  brown  colonies. 
It  inverts  and  ferments  saccharose. 

SCHIZOSACCHAROMYCES  APHALARAE  CALTHAE.    Sulc 

This  yeast  was  discovered  by  Karel  Sulc 3  in  the  larvae  of 
Aphalarae  calthae  (Homoptera).  It  possesses  spherical  cells  which  are 

1  Guilliermond,    A.      Remarques    sur   la   copulation   du   Schizosaccharomyces 
mellacei.     Bull,  de  la  Soc.  Botanique  de  Lyon,  April,  1903:    Thesis  for  the  Doc- 
torate mentioned  elsewhere  in  this  volume. 

2  Eykmann,    C.      Mikrobiologisches    iiber    die    Arrakfabrikation    in    Batavia. 
Cent.  Bakt.  16,  1894. 

3  Sulc,    K.      Pseudovitellius    und    ahnliche    Gewerbe    der    Homopteren    sind 
wohnstatten  symbiotischer  Saccharomyceten.     Sitzungsberichte  der  Konig.  Bohm. 
Gesellsch.  der  Wessinschaften  in  Prag.     March  30,  1910. 


SCHIZOSACCHAROMYCES   FORMOSENSIS 


203 


short  (about  1  to  2  /z  in  length)  containg  a  nucleus  and  metachromatic 
granules.     The  cells  are  often  grouped  in  twos.     (Fig.  80,  1  to  4.) 

SCHIZOSACCHAROMYCES  FORMOSENSIS.     Nakazawa  * 

This  species  was  isolated  recently  by  Nakazawa  from  sugar  prod- 
ucts in  Formosa.     On  beer  wort,  the  cells  are  ellipsoidal  or  irregular 


Fig.  80.  —  Sch.  Aphalarae  Calthae. 

1  and  2,  Vegetative  cells;  3,  two 
gametes  about  to  unite  (?);  4,  egg 
resulting  from  the  fusion  of  the  two 
gametes  (?);  5,  6,  cells  dividing  by 
budding;  7-9,  cells  dividing  by  parti- 
tion; 10-12,  beginning  of  germination 
of  the  ascospores  (after  Sulc). 


Fig.     80-A.  —  Schizosaccharomyces     For" 
mosensis,  var.    Tapaniensis. 


b,    Copulation;     d,    ascs; 


;     /, 
r  Na 


f1,    germination    of 


in  shape  (9.2-16.8  X  4.8 /z  but  usually  about  7.2  X  6-9  /*).  The 
optimum  temperature  for  budding  in  beer  wort  is  32°  C.  The  ascs 
are  formed  at  the  end  of  7  days  and  are  derived  from  an  isogamic 
copulation  similar  to  those  in  Schizosac- 
charomyces Pombe.  They  contain  ellipsoi- 
dal ascospores  with  no  glycogen,  but 
their  walls  are  impregnated  with  starch. 
On  wort,  a  ring  is  formed  at  25  to  37°  C. 
and  at  32°  C.,  but  not  below.  At  25  to 
27°  C.,  a  scum  is  formed.  The  temperature 
limits  for  scum  formation  are  25  to  27°  C. 
and  37°  C.  This  yeast  ferments  dextrose, 

inuline,  dextrine,  d-mannose,  d-galactose,  Fig  8'o_B-_  Schizosaccharomyces 
d-fructose,  saccharose,   maltose,  raffinose,    Formosensis.  Vegetative  Cells, 
and  a-methylglucoside.  Foofmatspore0sf  %£?'  "SiSS^^ 

Nakazawa  described  two  other  varieties     Jef  t|£^S  &£^"*^**'' 
of    this    yeast,    Schizosaccharomyces 

Formosensis  and  Schizosaccharomyces  Formensis,  var.  akoensis.  This 
latter  variety  differs  from  the  former  by  larger  cells  and  larger 
ascospores  and  also  that  the  ascs  are  formed  at  the  end  of  5  days. 
No  ring  is  produced  at  32°  C.  and  scums  are  not  produced  at  all. 
Schizosaccharomyces  Formosensis,  var.  tapaniensis,  another  variety, 

1  Nakazawa,  R.     Ueber  Gamngsmikroorganismen  von  Formosa  II.    Ber.  der 
Inst.  fur  Exper.  Forshungen,   1914. 


204 


FAMILY  OF  SACCHAROMYCETACEAE 


has  cells  intermediate  between  the  two  previous  species,  forms  its 
ascs  at  the  end  of  four  days  and  produces  a  ring  at  32°  C.,  but  never 
forms  a  scum. 


SCHIZOSACCHAROMYCES  SAUTAWENSIS.     Nakazawa 

This  species  was  isolated  by  Nakazawa  from  sugar  in  Formosa.    It 
has  elliptical  cells  when  grown  in  beer  wort  (7.2—8.4  X  4.8  usually, 

rarely  7.2- 19.2  X  4.8  jx).  The 
optimum  temperature  for  growth 
on  beer  wort  is  32°  C.  Ascs 


Fig.  80-C.  —  Schizosac- 
charomyces sautawensis. 
Vegetative  Cells. 

6,  c,  and  h,  Copulation ;  d,  ascs ;  /,  f1, 
germination  of  spores  (after  Naka- 
zawa). 


Fig.  80-D. — Schizosaccharomyces 
formosensis. 

6,  c,  c1,  Copulation  of  the  asc;  /,  germi- 
nation of  spores  (after  Nakazawa). 


are  formed  at  the  end  of  seven  days.  They  are  derived  from 
an  isogamic  copulation,  possessing  ellipsoidal  ascospores  without 
glycogen  and  with  starchy  walls  (2.5  X  3  to  3.75  ju).  A  ring  is 

formed  at  from  25°  to  32°  C.,  but 
none  is  produced  at  37°  C.  It 
gives  a  scum  at  from  25  to  27°  C. 
It  ferments  dextrine,  dextrose, 
d-mannose,  d-galactose,  d-fructose, 
saccharose,  maltose,  raffinose  and 
a-methylgluco^ide. 

SACCHAROMYCES  NOK- 
KOENSIS.    Nakazawa 

Isolated   under   the   same   con- 

8(>_E.  _  Schizosaccharomyces  ditions  as  the  preceding  type,  this 
Nokkoensis.  yeast  possesses-  ellipsoidal  cells  on 

spores;  beer  wort  (9.6-10.8  X  3.6-5.25, 
rarely  4.8-16.6  X  3.6-5.2  /x).  The 
optimum  temperature  for  growth  in  beer  wort  is  32°  C.  Ascs  are 
formed  after  two  months,  without  a  copulation.  The  ascospores  are 
ellipsoidal,  spherical,  or  hemispherical  (3X5  microns)  without  glycogen 
and  with  starchy  walls.  A  ring  is  produced  on  beer  wort  at  25-32°  C. 


a,    Vegetative    cells;    6,     germinating 
g,  ascs  (after  Nakazawa). 


SCHIZOSACCHAROMYCES  CHERMETIS  ABIETIS     205 

but  more  at  37°.     No  scum  is  formed.     It  ferments  dextrine,  dextrose, 
d-mannose,  d-galactose,  d-fructose,  saccharose,  maltose  and  raffinose. 

SCHIZOSACCHAROMYCES   CHERMETIS  ABIETIS.   Sulc 

This  yeast  was  found  in  Chermes  abietis  and  resembles  Sch.  Pombe 
very  much.  The  cells  are  oval.  They  were  not  cultivated  by  Sulc. 
In  the  larvae  of  Psylla  Foersteri  he  found  Sch.  Aphidis;  Sch.  Psyllae 
Foersteri  was  found  in  the  larvae  of  various  homoptera.  He  also  ob- 
served in  certain  of  the 
homoptera,  fungi  resem-  ifj  A 

bling  the    Schizosaccharo-     B  \ 

myces  in  which   multipli-      "  ^ 

cation   was    accomplished       l  2 

by    division    or    budding 
and  which  did  not  form  4 

ascospores.       The     author      Fig.  81.  —  Sch.  Chermetis  strobilobii  (1  to  4)  and 
gave    them    the    generic        Sch'  Chermeiis  abietis  (5  and  6)  (after  Sulc). 
name  of  Cicadomyces.      These  are  distinguished  from  the  Schizosac- 
charomyces  by  the  fact  that  their  division  remained  for  a  long  time 
incomplete;   the  cells  remain  united  at  their  apexes  and  are  able  to 
form  chains  of  cells.     The  author  describes  C.  Ptyeli  lineati  and  the 
C.  Aphalarae  calthae. 

Hollande  l  has  observed  the  yeast  forms  in  the  blood  of  other 
insects.  He  studied  the  blood  of  the  locust  (Caloptenus  italicus). 
Under  normal  conditions,  the  blood  of  this  insect  is  yellow,  but  when 
it  is  infected  with  the  yeast  it  is  a  milky  white.  Hollande  could  re- 
produce the  infection  only  by  injecting  blood  from  an  infected  insect 
into  a  healthy  insect.  These  insects  died  i  from  5  to  7  days  and 
their  blood  was  found  to  be  fired  wit  the  yeast  parasite. 

The  yeast  structures  were  cylindrical.  Their  dimensions  varied 
from  4.98  to  6.64  microns  in  length  and  from  1.70  to  2  microns  in 
width.  A  vacuole  is  present  in  each  end  of  the  cell.  Buds  may 
appear  at  the  end.  After  staining  with  ferric  hematoxyline  a  cir- 
cular nucleus  is  observed  which  is  rich  in  chromatin.  This  parasite 
grows  well  on  blood  serum  with  a  white  scum;  on  gelatin,  growth 
is  abundant.  Fine  filamentous  structures  may  be  seen  at  the  edge  of 
the  cells.  No  spores  could  be  demonstrated. 

SECOND  GROUP 

Yeasts  multiplying  by  budding,  and  in  which  the  ascs  are  derived 
from  a  copulation,  or  show  traces  of  sexuality  in  their  origin. 

1  Hollande,  A.  Ch.  Formes  levures  pathogenes  observee  dans  le  sang  d'Acri- 
dium  (Caloptenus  italicus).  Comp.  Rend.  Acad.  Sci.  168  (1919),  1341-1344. 


206  FAMILY  OF  SACCHAROMYCETACEAE 

Genus  II.     Zygosaccharomyces.     Barker 

Ascs  resulting  from  a  copulation  of  two  cells.  Ascospores  in  a 
membrane  which  is  smooth. 

ZYGOSACCHAROMYCES  BARKER!.    (Barker)  Saccardo-Sydow  ' 

This  species  was  found  by  Barker  2  in  ginger  beer  to  which  had 
been  added  saccharose  and  nutrient  salts.  They  have  the  shapes  of 
small  oval  cells.  (Fig.  82.)  The  maximum  temperature  for  budding 

on  nutrient  gelatin  is  in  the  vicinity  of  37- 
38°  C.;  the  minimum  near  10-13°  C. 

The  ascospores  appear  easily,  not  only  on 
plaster  blocks  but  in  great  numbers  on  other 
media  (nutrient  gelatin,  damp  bread,  potato, 
carrot).  The  maximum  temperature  for 

the  formation  of  ascospores  on  plaster  blocks 
Fig.     82.  —  Zygosaccharomy-    .  .  f 

ces    Barkeri.      Vegetative  is  37-38     C.,  the  minimum   around  13  .    At 


Barker)^     ASCS     ^'^   25~27°    the    first    rudiments    of    ascospores 

appear  at  the  end  of  20  to  24  hours. 

The  ascs  result  from  an  isogamic  copulation  between  two  cells. 
This  copulation,  which  has  been  described  by  Barker,  is  accomplished 
in  the  same  manner  as  in  Sch.  Pombe  and  mellacei.  Two  cells  iden- 
tical in  characteristics,  unite  by  means  of  a  copulation  canal  formed  by 
the  fusion  of  a  little  projection  from  each  cell.  The  fusion  remains 
incomplete  and  the  cells  look  like  a  dumb-bell.  The  ascospores  are 
formed  in  the  swelled  portions  of  the  asc.  Their  number  varies  be- 
tween two  and  four.  (Fig.  82.)  Zygosaccharomyces  Barken  does  not 
form  a  scum  on  sugar  solutions,  but  at  the  end  of  10  to  14  days  a  ring, 
made  up  of  oval  cells,  appears.  It  ferments  dextrose,  levulose,  sac- 
charose, and  a-methylglucosides  but  neither  maltose,  lactose  nor  dextrine. 

ZYGOSACCHAROMYCES  PRIORIANUS.     Klocker  3 

This  spscies,  recovered  by  Klocker  from  the  bodies  of  bees,  pos- 
sesses cells  of  varying  shapes,  elongated,  round  or  oval,  sometimes  in  the 
shape  of  a  sausage  and  almost  always  united.  The  temperature  limits 
of  budding  are:  maximum,  36-38°  C.,  and  minimum,  3—8°  C. 

Spores  are  easily  produced  on  gelatin  or  wort,  carrot  or  agar. 
On  plaster  blocks,  on  the  contrary,  they  form  very  slowly.  The  limits 
for  the  formation  of  ascospores  on  plaster  blocks  saturated  with  beer 
wort,  are  27-28°  and  3-9°  C. 

1  Saccardo  and  Sydow.     Syllage  fungorum,  Vol.  II,  1902. 

2  Barker,  P.     A  conjugating  yeast  (Zygosaccharomyces  n.  gen.).       Proc.  of 
the  Royal  Society,  Vol.  6,   July  8,   1901.     On  the  spore  formation  among  the 
Saccharomycetes.    Journal  of  the  Federate  Institutes  of  Brewing,  8,  1902. 

3  Klocker.     In  Lafar's  Handbuch  der  technischen  Mykologie,  Jena,  1904-1905. 


ZYGOSACCHAROMYCES  JAPONICUS  207 

Klocker  showed  in  1904  that  the  ascs  of  this  yeast  are  derived 
from  an  isogamic  copulation;  consequently  it  is  related  to  the  genus 
Zygosaccharomyces.  Copulation  is  accomplished  exactly  as  in  Zy. 
Barken.  The  ascs  are  formed  of  two  large  portions  united  by  a  canal 
(Fig.  83).  The  ascospores  in  the  number  of  2  or  4 
are  formed  in  each  large  part  of  the  asc;  they  are 
round  or  oval.  We  have  shown  that  quite  a  few  of 
the  cells  form  ascospores  without  having  undergone 
copulation.  Parthenogenesis  is,  then,  rather  fre- 
quent. (Fig.  83,  a,  b,  c.)  This  yeast  forms  a  rather 

scant  scum  but  very  often  a  well-developed  ring.   Fig  83 Formation 

The  appearance  of  the  colonies  on  gelatin  at  the      of  the  Asc  in  Zyg. 
temperature  of  the  laboratory  resembles  the  shape      Pnonarms. 
a   cupule.     This   yeast  ferments  dextrose,   levulose,   saccharose  and 
maltose  but  not  lactose. 

ZYGOSACCHAROMYCES   JAVANICUS.     de  Kruyff 

This  species  was  isolated  in  Java  by  de  Kruyff  l  in  1908  on  partly 
decomposed  foliage.  It  is  a  yeast  with  elliptical  cells  from  4  to  8 
JJL  in  diameter.  The  optimum  temperature  for  budding  is  situated 
between  34  and  35°  C.;  the  maximum  is  around  38°.  Spores  appear 
easily  on  gelatin  and  are  preceded  by  an  isogamic  copulation.  Zy. 
javanicus  does  not  form  a  scum.  It  is  a  bottom  yeast  which  ferments 
dextrose,  levulose,  saccharose  and  d-galactose. 

ZYGOSACCHAROMYCES  JAPONICUS.    Saito 
Syn.:    SOYA-KAHMHEFE    Saito 

This  yeast  was  discovered  by  Saito  2  in  1906  among  the  products 
of  fermentation  of  Soya;  it  was,  at  first,  given  the  provisional  name 
of  Soya-Kahmhefe.  It  is  a  yeast  with  round  or 
°val  cells  (Fig.  84)  which  frequently  gives  long 
threads  made  up  of  budding  units.  The  ascs  in 
certain  numbers  are  observed  in  the  scum  grow- 
ing  on  Koji  or  in  cells  developing  on  gelatin. 
They  are  easily  obtained  on  Gorodkowa's  gelatin 
medium.  Saito  in  1909  showed  that  they  were 

.  84-  ~  zy%  Jap™-  preceded  by  a  copulation  similar  to  that  of  Zyg. 
icus  (after  Saito).  * 

Barken   and  on  account   of  this,   this  yeast  is 

attached  to  the  Zygosaccharomyces.     The  ascs  resemble  retorts  united 

1  de   Kruyff,   E.     Untersuchungen   iiber  auf    Java   einheimische   Hefenarten. 
Cent.  Bakt.  21,  1908. 

2  Saito,  K.    Mikrobiologische  Studien  liber  Soyabereitung.     Cent.  Bakt.  17,  1906. 


208  FAMILY  OF  SACCHAROMYCETACEAE 

by  their  necks.  (Fig.  85.)  The  number  of  ascospores  varies  in  each 
asc.  They  are  spherical  (2.7  to  6.3  /x  in  diameter)  and  are  relatively 
resistant.  Germination  is  accomplished  by  budding.  Guilliermond 
has  noticed  frequently  cases  of  parthenogenesis  in  this  yeast  in  which 
the  ascs  form  without  preliminary  copulation.  On  decoction  of  "  Koji," 

-<o  o&°     ^s  yeas^  Pr°duce3  a  white  farinaceous  scum. 

*  c*t  J^  On  the  surface  of  this  scum  a  number  of  bub- 
bles  of  carbon  dioxide  form.  The  scum  increases 


slowly  and  turns  to  a  light  brown. 

The  giant  colonies  are  raised,  dry  and  light 
®  §     gray  in  color.      The   surface   is   concentrically 
Fig.  85.—  Copulation  and   ringed  and  cut  up  by  fissures.     The  edge  of  the 
Formation  of  the  Ascs   colony  is  notched.     Under  the  colony  numerous 
in  Zyg.  Japonic. 


The  cultures  on  gelatin  have  a  light  brown  appearance.  This 
species  ferments  dextrose,  levulose  and  maltose  but  has  no  action  on 
6-galactose,  lactose,  saccharose,  melibiose,  raffinose,  a-methyl-gluco- 
side  and  inuline.  Zyg.  japonicus  by  the  character  of  its  scum,  ap- 
proaches the  genera  Willia  and  Pichia  very  much.  It  is  distinguished, 
however,  by  the  fact  that  the  scum  completely  developes  only  in 
decoctions  of  "Koji."  Takahashi  and  Yukawa1  have  recently  re- 
described  this  yeast  as  follows: 

This  species  was  isolated  from  many  samples.  Since  this  yeast 
and  Zygosaccharomyces  salsus  develop  and  form  particular  grayish 
brown  films  even  on  a  "  Shojii  "  which  any  other  kinds  of  film-form- 
ing yeast  could  not  grow,  both  these  yeasts  are  feared  in  storing 
"  Shoju."  Moreover,  this  species  very  easily  forms  large  numbers 
of  sporulated  cells. 

Young  cells  from  the  surface  cultures  on  "  Koji  "-extract  agar 
are  round  (commonly  4-8  c.c.)  or  oval,  and  contain  glycogen.  In 
old  cultures  club-shaped  or  mycelial  cells  are  often  observed.  Most 
of  the  cells  in  a  diluted  "  Shoju  "  are  elongated  abnormally  and  in- 
crease the  number  of  vacuoles. 

On  plate  culture  of  wort  gelatin,  this  yeast  forms  a  grayish  white, 
crater-like,  elevated  colony  with  smooth  periphery,  and  the  color 
turns  brownish  after  the  lapse  of  time.  On  "  Koji  "-extract-gela- 
tin streak  culture,  the  growth  shows  a  grayish  white,  somewhat 
dried,  lustered,  folded  covering  with  fine  toothed  edge.  On  "  Koji  "- 
extract  culture  at  23°  C.  it  forms  mealy  white,  small  filmy  fragments 
on  the  surface,  and  covers  the  whole  surface  after  3  days.  The  film 

1  Takahashi,  and  Yukawa,  M.  On  the  budding  fungi  of  "Shoju-Moromi." 
Original  Communications  of  the  8th  International  Congress  of  Applied  Chemistry, 
14  (1912),  155-171. 


ZYGOSACCHAROMYCES  SOYA  209 

crinkles  like  crepe  paper,  increasing  its  thickness,  and  its  color  changes 
to  yellowish  brown.  After  three  weeks  the  film  gradually  falls  down 
and  deposits  a  great  deal  of  sediment  on  the  bottom,  leaving  a  thin 
film  over  the  surface,  and  at  last  only  a  few  parts  of  the  yeast  ring 
remain  along  the  wall.  In  wort  culture  at  23°  C.,  after  keeping  the 
culture  for  seven  days  film  does  not  form,  although  gas  bubbles  as- 
cend through  the  medium.  After  three  months  a  well-defined  yeast 
ring  and  thin  film  become  observable,  but  this  film  has  never  been 
folded  at  all.  This  species  reproduces  and  forms  its  particular  film 
even  in  "  Koji  "-extract  or  "  Shoju  "  which  contains  23%  of  NaCl. 

It  is  most  noticeable  that  this  species  forms  a  grayish  brown,  folded 
film  on  the  surface  of  sterilized  "  Shoju  "  after  a  long  time,  while  other 
races  which  we  isolated  from  "  Shoju-Moromi  "  cease  the  reproduction 
of  their  cells  in  the  same  medium. 

This  species  ferments  dextrose,  maltose,  levulose,  but  does  not 
ferment  saccharose,  lactose,  raffinose,  a-methylglucoside,  galactose. 

It  rarely  produces  spores  in  a  yeast  ring  of  "Koji  "-extract  cul- 
tures after  three  months.  After  Gorodkowa's  method,  sporulation 
occurs  often  after  10  days  at  28°  C.  Following  the  diluted  "  Shoju  " 
method  which  has  been  described  in  the  preceding  plate,  a  large  num- 
ber of  sporulated  cells  occur  in  the  yeast  ring  after  4-5  days.  The 
spore  is  transparent  with  a  somewhat  thick  wall;  it  is  round  or  oval 
in  shape,  2.5-6  fj,  in  size,  and  contains  a  few  tiny  grains.  The  proc- 
esses of  formation  and  germination  of  spores  in  this  yeast  and  other 
relations  are  similar  to  the  preceding  yeasts.  This  yeast  seems  to  be 
identical  with  Zygosaccharomyces  japonicus,  Saito. 

Mitsuda  and  Nishimura  have  found  in  the  fermentation  of  Soya 
an  asporogenic  yeast.  Kita  has  recently  described  a  yeast  similar  to 
Zygosaccharomyces  major  (Takahashi  and  Yukawa)  which  differs  in 
that  galactose  is  not  fermented. 

ZYGOSACCHAROMYCES   SOYA?      (Saito)  Guilliermond 

This  yeast  was  found  with  the  preceding  yeasts  by  Saito1  in  the 
products  from  the  preparation  of  Soya  sauce  and  designated  as  Sac- 
char omyces  Soya.  It  is  made  up  of  spherical  or  oval  cells  (4  to  8  ju, 
average  size)  with  relatively  resistant  walls  and  homogeneous  con- 
tents with  large  vacuoles.  (Fig.  86.)  No  ascospores  are  produced  on 
plaster  blocks,  or  in  yeast  water  to  which  dextrose  has  been  added 
or  pure  agar;  abundant  sporulation  is  observed,  however,  in  the  rings 
formed  on  a  decoction  of  Koji.  The  ascs  are  usually  made  up  of  two 

1  Saito,  K.  Mikrobiol.  Studien  uber  die  Soya-Bereitung.  Cent.  Bakt.  17, 
1906. 


210 


FAMILY  OF  SACCHAROMYCETACEAE 


swelled  parts  connected  by  a  canal  and  offer  a  resemblance  to  the 
ascs  of  the  Zygosaccharomyces  and  we  shall  not  hesitate  to  attach  this 
yeast  to  this  genus  although  copulation  has  not  been  observed  up 
to  the  present  time.  (Fig.  86.) 

The  ascospores  are  generally  to  the  number  of  from  1  to  4  per  asc 
and  are  located  in  each  part  of  it.  They  are 
spherical,  hyaline  and  possess  a  resistant  wall. 
Their  diameter  is  from  2.7  to  4.5  microns. 

Giant  colonies  are  a  grayish  brown  color; 
they  are  raised  in  the  center  and  have  a  greatly 
notched  edge.  On  plates,  colonies  are  produced 
which  resemble  dots,  round  and  moist.  On 
gelatin  streaks  or  stabs  the  yeast  offers  a  greenish 
Cells  and  KSa?  *€>?  coloration  along  the  line  of  inoculation  and  on 

Zygosaccharomyces  the  surface  a  grayish  yellow.     Zygosaccharomyces 
Soya  (after  Saito).  »  ,        ,  i        i  j 

soya    ferments    dextrose,    levulose,    d-mannose, 

d-galactose  and  maltose  easily.  It  does  not  act  on  saccharose,  inuline, 
a-methylglucoside,  lactose,  melibiose,  or  raffinose.  It  inverts  sac- 
charose but  does  not  ferment  it.  Its  invertase  does  not  pass  through 
the  cell  wall  and  is,  therefore,  an  endoenzyme. 

ZYGOSACCHAROMYCES  LACTIS   a.   W.   Dombrowski 

This  species  isolated  by  Professor  Jensen  from  beer  has  been 
described  by  Dombrowski 1  (1910).  On  beer  wort  the  cells  are  spheri- 
cal and  have  an  average  diameter  of  4.7  JJL.  Sporulation  is  preceded 
by  an  isogamic  copulation  which  is 
effected  after  the  normal  procedure. 
The  ascs  which  result  are  made  up  of 
two  enlarged  portions  connected  by  a 
canal.  (Fig.  87,  2  and  3.)  The  asco- 
spores are  formed  in  these  two  enlarged 
parts  and  vary  from  one  to  four  in  each 
asc.  Germination  is  accomplished  by  an 
absorption  of  the  wall  of  the  asc  after 
which  ordinary  budding  is  accomplished. 
This  species  produces,  on  beer  wort  at 
room  temperature,  a  scant  scum  made 
up  of  cells  with  a  normal  shape. 

The  colonies  on  plates  are  lenticular 
and  are  round  or  torpedo  shaped.    In  stab  cultures  the  growth  extends 
about  3.5  centimeters  below  the  surface.     Giant  colonies  offer  a  crater- 


Fig.  87.  —  Zyg.  Lactis  a. 


Vegetative  cells.  —  2  and  3.  Ascs  (after 
Dombrowski). 


1  Dombrowski,  W. 
28,  1910. 


Die  Hefen  in.  Milch  und  Milchprodukten.     Cent.  Bakt. 


ZYGOSACCHAROMYCES  BAILII 


211 


iform  center  about  which  there  is  a  sort  of  raised  wall.  The  edge  is 
slightly  fringed  and  partly  covered  with  ridges.  It  produces  an  energetic 
fermentation  in  beer  wort  which  it  clears  in  about  10  days.  The  wort 
is  slightly  colored  and  made  to  give  off  an  aromatic  odor.  In  about 
100  c.c.  of  wort,  after  five  and  a  half  months,  it  forms  about  4.5 
grams  of  alcohol.  Zygosaccharomyces  lactis  a  produces  an  active 
fermentation  in  milk.  It  ferments  lactose,  saccharose  and  dextrose 
and  d-galactose  but  has  no  action  on  maltose.  Along  with  the  alcohol 
and  carbon  dioxide  which  it  produces,  small  quantities  of  acids  are 
produced. 

ZYGOSACCHAROMYCES  BAILII    (?)    (Lindner)   Guilliermond 
Syn.     SACCHAROMYCES  BAILII.     Lindner 

This  yeast  was  isolated  from  beer  by  Lindner.1  The  cells  are  large 
and  elongated,  and  have  resistant  walls.  The  contents  are  ordinarily 
homogeneous  and  brilliant.  In  old  cultures 
they  possess  a  peculiar  irregular  ameboid  shape. 
At  the  end  of  a  long  series  of  cultures  on 
gelatin,  this  yeast  loses  its  property  of  sporulat- 
ing.  The  ascospores  are  very  refractive  almost 
devoid  of  granules  and  resistant  walls.  A  copu- 
lation seems  to  be  indicated  by  the  shape  of 
the  asc  which  is  made  up  of  two  enlarged  parts. 
(Fig.  88.)  Then  it  is  about  certain  that  this  Fig.  88.  —  Saccharomyces 
species  may  be  incorporated  in  the  genus  Zy-  Lbdner)(aCCOrding  t0 

gosaccharomyces. 

This  opinion  is  confirmed  by  the  presence 
of  ameboid  cells  in  old  cultures,  some 
of  which  sporulate  and  which  seem  to 
be  endowed  with  unfruitful  attempts 
at  copulation.  No  scum  is  formed  by 
this  yeast;  on  hop  wort,  it  develops  at 
the  bottom  of  the  culture.  Fermenta- 
tion is  very  feeble  and  growth  less 
abundant.  The  must  gives  off  an  aro- 
matic odor. 

Giant  colonies  on  gelatin  or  beer 
wort  have  a  slow  growth  and  remain 
small.  The  surface  is  glistening  and  a 
grayish  white.  Gelatin  is  not  liquefied.  The  cells  often  present  an 
ameboid  shape. 

1  Lindner,  P.  S.  farinosus  et  Bailii.     Woch.  Brau.     1893. 


Fig.  88-A.  —  Zygosaccharomyces 
Mandshuricus.  Sporogenic  Race 
from  Sediment  in  Beer  Wort  at 
28°  C.  (after  Saito). 


212 


FAMILY   OF  SACCHAROMYCETACEAE 


On  gelatin  streaks,  a  whitish  deposit  forms.  In  gelatin  stabs 
the  yeast  shows  a  tendency  to  form  lateral  rays.  In  gelatin  to  which 
must  has  been  added,  vesicles  of  carbon  dioxide  are  formed  here  and 
there.  This  yeast  is  able  to  ferment  only  dextrose  and  levulose. 


ZYGOSACCHAROMYCES  MANDSHURICUS.    Saito1 

This  yeast  was  isolated  from  Chinese  yeast  which  is  used  to  pre- 
pare the  "  1'eau  de  vie  "  in  Manchuria,  aft  alcoholic  drink  known  as 
Sorgho.  The  cells  are  round  or  oval  (6.5-9.5  JJL  in  diameter).  The 
giant  colonies  are  round  or  oval  with  a  flat  edge.  On  plaster  blocks 


Fig.  88-B. — Zygosaccharomyces 
Mandshuricus  (after  Saito). 
Copulation  and  Formation  of 
the  Ascs. 


Fig.  88-C.  —  Zygosac- 
charomyces Mand- 
shuricus. Partheno- 
genetic  Ascs  from 
Cells  of  a  White 
Race  (after  Saito). 


Fig.  88-D.  —  Zygosac- 
charomyces Mand- 
shuricus. Asporo- 
genic  Variety.  Cells 
from  Sedimental 
Growth  in  Wort  at 
20°  C.  (after  Saito). 


in  beer  wort,  and  on  Gorodkowa's  medium,  ascs  are  formed  con- 
taining one  to  four  spores  (45  ju  in  diameter).  These  result  from 
an  isogamic  copulation.  The  temperature  limits  for  sporulation  are 
30  to  35°  C.  This  yeast  ferments  dextrose,  levulose,  and  mannose 
energetically,  saccharose  feebly. 


ZYGOSACCHAROMYCES  MELLIS  ACIDI.     Richter 

Isolated  from  honey  undergoing  an  alcoholic  fermentation,  this 
yeast  possesses  cells  of  small  dimensions.  The  ascs  are  formed  by 
isogamic  copulation.  The  yeast  ferments  glucose,  fructose  and  sac- 
charose actively  and  galactose  feebly,  but  has  no  action  on  other 
sugars. 

1  Saito,  K.  Mikrobiologische  Studien  iiber  die  Bereitung  des  Mandschurischen 
Branntweins.  Report  of  the  Central  Laboratory,  South  Manchuria  Railway  Co. 
1914.  Bull.  Past.  Inst.  12  (1915)  1. 


ZYGOSACCHAROMYCES  NADSONII  213 


ZYGOSACCHAROMYCES  NADSONII.     Guilliermond 

This  species  was  isolated  by  Guilliermond  l  from  a  large  bottle 
of  orange  syrup  at  Hospital  101  at  Lyon  during  the  summer  of  1915. 
In  this  it  caused  a  very  active  fermentation.  After  12  hours  in  beer 
wort  at  25-30°  C.,  this  yeast  forms  a  white  deposit  at  the  bottom 
of  the  flask.  In  a  few  days  there  is  produced  over  the  surface  of 
the  liquid  a  very  delicate  covering.  Later  a  ring  develops  which 
falls  to  the  bottom  of  the  flask  when  it  is  disturbed.  This  ring 
does  not  seem  to  re-form.  The  cells  are  generally  round  or  oval  in 
beer  wort  cultures  which  are  12  hours  old. 
Sometimes  they  are  isolated  but  generally 
they  adhere  in  a  small  group;  after  24 
hours  this  tendency  is  greatly  increased. 
The  cells  are  generally  round  or  oval  and 

form  buds  around  their  peripheries.      The  Fig-   88-E.    —  Zygosaccharo- 

,,  11      ,T  e   m       7          A^  myces    chevalien.      1,    Cells 

cells  may  resemble  those  of  Torula.    After      from  Sediment  in  Beer  Wort 

8  to  14  hours  the  elongated  cells  may  be-       24  Hours  at  25°  C.;  2,  Asco- 

come  rather  numerous.     On  must  agar,  the 

colonies  develop  quickly;   on  carrot,  a  white  blanket  growth  is  formed. 

The  minimum  temperature  seems  to  be  situated  between  3  and  5°  C. 
At  5°  C.  and  up  to  15°  C.  the  yeast  develops  slowly.  At  18°  C.  and  20°  C. 
its  development  becomes  more  rapid.  The  optimum  temperature  is 
situated  near  30°  C.  and  the  maximum  is  between  41°  C.  and  42°  C. 
In  the  vicinity  of  these  temperatures  the  yeast  shows  a  tendency 
to  elongate  and  form  cells  like  the  Pastorianus  type. 

The  ascospores  form  very  abundantly  after  a  few  days  on  Gorod- 
kowa's  media.  They  seem  to  be  formed  in  great  numbers  but  less 
rapidly  on  carrot  and  beer  wort  agar.  The  ascs  result  almost  uni- 
versally from  a  heterogamic  conjugation.  It  is  very  easy  to  follow 
all  the  stages  in  this  phenomena  in  a  culture  on  Gorodkowa's  agar. 
At  the  moment  when  conjugation  is  about  to  occur  most  of  the  cells 
affected  show  in  their  center  a  large  number  of  small  fat  globules. 
Almost  all  are  united  in  small  colonies  made  up  of  a  small  number 
of  cells.  Conjugation  is  effected  between  a  mother  cell  and  an  in- 
completely developed  daughter  cell.  The  mother  cell  plays  the  role 
of  a  female  gamete  while  the  daughter  cell  represents  the  male  gam- 
ete. The  gametes  are  then  represented  by  cells  of  different  ages. 
The  male  gamete  (microgamete)  is  a  cell  which  has  not  achieved  its 
full  growth,  while  the  female  gamete  (macrogamete)  is  full  grown. 

1  Guilliermond,  A.  Zygosaccharomyces  Nadsonii.  Nouvelle  espece  de  levures 
a  conjugaison  he"te"rogamique.  Bull.  Soc.  de  France,  34  (1918). 


214  FAMILY  OF  SACCHAROMYCETACEAE 

The  two  gametes  unite  by  a  copulation  canal  formed  by  the  union 
of  two  small  projections  sent  out  from  each  cell.  During  this  phenom- 
enon the  small  cell  remains  adherent  to  the  mother  cell  and  after 
the  development  of  the  copulation  canal  may  detach  from  this  cell. 
The  contents  of  the  male  gamete  emigrate  to  the  female  gamete, 
both  protoplasms  fuse,  and  the  female  gamete  is  transformed  into  an 
egg;  this  changes  very  quickly  into  an  asc,  and  each  asc  contains 
usually  from  one  to  two  ascospores;  exceptionally  one  may  find 
three,  or  even  four. 

Besides  this  heterogamic  conjugation  which  has  been  described, 
frequently  one  may  observe  a  series  of  transitional  forms  between  the 
iso-  and  heterogamic.  The  conjugation  presents  the  same  characteris- 
tics in  cultures  on  carrot  and  beer  wort.  However,  in  the  latter  me- 
dium the  yeast  seems  to  take  on  the  elongated  form. 

The  minimum  temperature  for  sporulation  on  Gorodkowa's  agar 
seems  to  be  situated  around  5°.  At  this  temperature  the  first  rudi- 
ments of  ascospores  appear  at  the  end  of  two  weeks;  at  from  13-15° 
the  ascospores  form  at  the  end  of  eight  days;  from  19-20°  they 
appear  at  the  end  of  56  hours;  the  optimum  temperature  seems  to 
rest  between  23  and  30°,  while  the  maximum  temperature  is  situated 
somewhere  between  30  and  32°.  The  ascospores  remain  enclosed  in 
the  wall  of  the  asc.  During  the  first  stages  in  germination,  the  wall 
is  ruptured,  and  the  ascospores  germinate  by  ordinary  budding. 

In  plate  cultures  the  colony  is  of  yellowish  white  with  a  peripheral 
zone  made  up  of  canals;  the  center  is  a  little  elevated  and  is  con- 
structed of  rather  thick,  irregular  folds.  In  streak  cultures  the  colony 
presents  the  same  characteristics.  In  stab  cultures  the  yeast  develops 
abundantly  along  the  line  of  inoculation  and  forms  a  large  number 
of  small  colonies.  The  colonies  enlarge  toward  the  surface.  The 
giant  colonies  on  must  gelatin  at  from  15-20°  have  a  dry  appearance 
and  are  only  a  few  millimeters  in  diameter.  The  color  is  yellowish 
white,  the  center  is  slightly  raised, 

This  yeast  inverts  saccharose  when  cultivated  in  must.  It  forms 
carbon  dioxide  which  gives  evidence  of  fermentation.  This  fermen- 
tation is  well  demonstrated  by  Lindner's  droplet  culture  method. 
According  to  this  method  the  yeast  ferments  dextrose  and  levulose. 
It  has  no  action  on  lactose,  d-galactose,  maltose,  dextrin  and  raffinose. 

The  existence  of  a  heterogamic  conjugation  is  of  special  biological 
interest,  because  heterogamy  up  to  the  present  time,  has  been  rarely 
observed  among  yeasts.  Guilliermond  found  it  in  the  yeasts  which 
he  named  Saccharomyces  chevalieri.  In  this  same  paper  Guilliermond 
has  given  a  rather  extended  discussion  on  this  subject  in  its  relations 
to  the  yeasts.  -  -*/ 


ZYGOSACCHAROMYCES  MAJOR  215 

ZYGOSACCHAROMYCES  MAJOR.     Takahashi  and  M.  Yukawa l 

This  yeast  was  isolated  from  samples  taken  at  different  stages 
during  the  ripening  of  "  Shoju." 

In  "Koji  "-extract  or  wort  culture  after  4  days  at  20°  C.,  the  cells 
are  mainly  spherical  (3.7-7.5  ju),  sometimes  oval,  their  contents  are 
homogeneous,  and  sometimes  exhibit  vacuoles.  The  glycogen  reac- 
tion is  evident  in  every  cell.  Cells  in  yeast  ring  of  "  Koji  "-extract 
culture  after  20  days  at  20°  C.  are  so  irregular  that  a  cell  may  be  as 
small  as  2.5  microns  and  as  large  as  10  microns.  The  occurrence  of 
these  cells  seems  to  be  somewhat  prolonged  in  wort  or  "  Koji " 
extract  containing  a  quantity  of  salt.  Old  culture  in  the  same 
media  after  2-6  months  at  room  temperature  exhibits  not  only  the 
cells  which  are  similar  to  Will's  film  cells  of  the  first  generation,  and 
permanent  cells,  but  also  very  highly  elongated,  mycelial  ones. 

In  "  Koji  "-extract  or  wort  gelatine  plate  after  7  days  at  room 
temperature  this  species  forms  grayish  white,  round,  bright,  waxy 
colonies.  On  "  Koji  "-extract-agar  plate  after  30  days  at  27°  C.  it 
grows  with  somewhat  brownish,  waxy,  dull  lustered,  elevated  cover- 
ing. Margin  shows  somewhat  paralleled  streamy  canals.  On  glu- 
cose-sake-agar  after  10  days  at  25°  C.  it  forms  a  grayish  white,  waxy 
covering  with  slightly  elevated  sides.  The  central  part  is  somewhat 
concaved  and  the  marginal  part  dull  toothed.  On  "  Koji  "-extract 
gelatin  stab  after  30  days  at  15°  C.  it  forms  waxy,  feeble  lustered, 
brownish,  elevated  isles  at  the  mouth  of  the  stab  canal,  and  rosary- 
like  colonies  with  gas  bubbles  along  the  canal.  This  species  grows 
in  many  fluid  media,  and  according  to  the  appearance  of  its  fermenta- 
tion it  belongs  to  the  so-called  bottom  yeasts.  In  "  Koji  "-extract 
culture  at  25°  C.  a  yeast  ring  appears  first  after  3  days,  but  it  does 
not  form  any  complete  ring  even  after  6  months,  -while  the  sedimental 
yeast  crop  becomes  somewhat  plentiful  after  3  weeks.  Its  develop- 
ment in  wort  or  hopped  wort  seems  to  be  inferior  to  that  in  "Koji  " 
extract.  Its  resisting  power  against  NaCl  is  so  striking  that  it  can 
grow  tolerably  in  "  Koji  "  extract  or  wort  containing  20%  NaCl. 

According  to  Lindner's  method  this  species  ferments  dextrose, 
levulose,  mannose,  saccharose,  maltose,  but  does  not  ferment  galac- 
tose,  lactose,  raffinose,  a-methyl-glucoside. 

This  species  is  one  of  easily  sporulable  kind  among  all  the  Zygo- 
saccharomyces  isolated  from  "  Shoju-Moromi."  This  yeast  does  not 
form  spores  on  gypsum-block  at  all.  Sporulated  cells  occur  very 
rarely  in  yeast  ring  developed  in  "  Koji  "-extract  culture  after  3  to  6 

1  Takahashi,  and  Yukawa,  M.  Original  communications,  Eighth  Internatl. 
Congress  of  ApDlied  Chemistry,  V.  XIV,  1912,  p.  162. 


216  FAMILY   OF  SACCHAROMYCETACEAE 

months  at  20°  C.  or  on  Gorodkowa's  agar  after  20  days  at  25°  C. 
On  the  other  hand,  following  the  diluted  "  Shoju  "  culture  which  has 
been  described  above  large  numbers  of  ascs  easily  occur  in  the  yeast 
ring  within  7-15  days.  The  processes  of  formation  and  germina- 
tion of  spores  are  similar  to  those  of  Zygosaccharomyces  soja  which 
have  already  been  described.  Spores  are  transparent,  round  or  oval, 
and  commonly  3-4.5  JJL  in  diameter.  A  few  tiny  grains  are  contained 
in  each  spore.  The  total  number  of  spores  in  each  asc  is  1-4;  but 
the  number  of  spores  which  occurs  in  each  part  is  quite  variable. 

This  species  seems  to  be  nearly  similar  to  Torula  "  Shoju  "  var. 
minuta  which  was  isolated  from  "  Shoju-Moromi  "  by  J.  Nichimura. 
It  is  necessary  to  ascertain  the  sporulation  of  the  latter  yeast  after 
our  method. 

This  yeast  differs  distinctly  from  Zygosaccharomyces  soja  and  as- 
porogenic  species  of  Zygosaccharomyces  by  the  following  characteris- 
tics: This  species  ferments  saccharose,  and  the  time  required  for 
sporulation  of  this  yeast  is  far  shorter,  and  the  number  of  sporogenic 
cells  in  yeast  ring  is  always  abundant. 

Zygosaccharomyces  salsus  distinguishes  itself  from  this  yeast  by  the 
formation  of  a  particular  film. 

ZYGOSACCHAROMYCES  CHEVALIERI.     Guilliermond 

This  yeast  was  isolated  from  products  of  fermentation  made  in 
Occidental  Africa  by  the  inhabitants  for  alcoholic  drinks.  They 
were  secured  through  the  Chevalier  Mission.  On  beer  wort  at  25°  C., 
there  is  formed  at  the  end  of  24  hours  an  abundant  sedimental  de- 
posit at  the  bottom  of  the  culture  flask  and  a  scum  not  containing 
air  but  with  a  grayish  and  slightly  viscous  appearance.  It  is  very 
delicate  and  falls  to  the  bottom  of  the  flask  when  it  is  disturbed.  An- 
other soon  re-forms.  The  cells  in  the  sedimental  deposit  are  variable 
in  shape,  sometimes  spherical,  usually  oval  or  ellipsoidal.  Others  are 
elongated.  Their  dimensions  vary  between  2-6  microns  long  and  4-8 
microns  wide.  Their  contents  are  transparent  with  a  vacuole  and 
many  brilliant  granules.  The  cells  are  generally  isolated  or  united  two 
by  two.  At  the  end  of  15  days  and  up  to  a  month,  the  cells  in  the 
sediment  show  a  tendency  to  elongate  and  remain  united  in  chains. 
One  may  find  5  to  10  elongated  cells  with  lateral  buds  and  branches 
which  make  up  a  sort  of  pseudomycelium.  The  temperature  limits 
for  budding  in  beer  wort  are:  maximum,  42-43°  C.;  minimum,  below 
5°  C.  Near  these  temperatures  this  yeast  does  not  develop  in  the 
form  of  a  sediment  nor  does  it  produce  a  scum.  The  cells  have  the 
same  shape  as  at  other  temperatures.  This  yeast  sporulates  easily 


ZYGOSACCHAROMYCES  CHEVALIERI 


217 


and  rapidly  on  most  solid  media,  such  as  slices  of  carrot,  gelatin  of 
Gorodkowa,  must  agar  and  gelatin.  Spores  are  also  formed  in  scums 
developed  on  beer  wort.  This  sporulation  is  preceded  by  a  hetero- 
gamic  copulation  which  has  been  described  before.1  This  copulation 
takes  place  between  two  gametes  of  variable  dimensions  and  of  dif- 
ferent ages.  One,  the  female  gamete  or  macrogamete  is  an  adult 
cell,  and  very  large;  the  other,  the  male  gamete,  or  microgamete,  is 
very  small,  and  young,  generally  a  bud  being  detached  from  a  mother 


Fig.  88-F.  —  Heterogamic  Copulation  in  Zygosaccharomyces 
Nadsonia. 

cell.  The  two  gametes  unite  by  a  copulation  canal,  then  the  con- 
tents of  the  microgamete  pass  into  the  macrogamete  which  becomes 
the  egg.  A  wall  is  formed  across  the  copulation  canal  which  separates 
the  egg  which  soon  appears  as  a  separate  cell.  This  changes  into  an 
asc  with  from  1  to  4  ascospores,  rarely  more.  By  their  form,  the 
spores  are  intermediate  between  those  of  the  genus  Pichia  and  of  the 
genus  Willia  but  they  approach  those  of  Pichia  most  closely.  Their 
dimensions  vary  between  1.5  and  2.5  jit. 

The  temperature  limits  for  sporulation  are:  maximum,  37-38°  C. 
and  the  minimum,  8-10°  C.  The  optimum  is  situated  near  25°  C.  to 
30  C.  The  spores  form  in  from  18  to  20  hours  at  this  temperature. 
Germination  of  the  spores  is  accomplished  by  ordinary  budding.  The 
spores  enlarge  and  lose  their  hemispherical  shape  when  budding. 

1  Guilliermond,  A.  Sur  un  example  de  copulation  heterogamique  observe 
chez  une  Levure.  Comp.  Rend.  Soc.  Biol.  1911. 


218  FAMILY  OF   SACCHAROMYCETACEAE 

The  giant  colony  on  must  agar  at  25°  C.  after  15  days,  is  well  de- 
veloped. The  color  is  gray,  slightly  yellow  with  a  dry  appearance. 
The  center  is  folded.  The  periphery  is  thin,  transparent,  and  pos- 
sesses a  border  made  up  of  fine  canals  with  deep  hollows.  At  the  end 
of  two  months,  the  colony  possesses  a  grayish  yellow  color  with  a  flat 
dry  appearance.  The  yeast  produces  no  fermentation  of  beer  wort. 
It  inverts  saccharose  but  yields  no  indication  of  fermentation  in 
saccharose,  dextrose,  levulose,  maltose,  d-mannose,  lactose,  d-galac- 
tose  and  dextrine. 

The  presence  of  a  scum  causes  this  yeast  to  form  a  sort  of  con- 
nection between  Hansen's  1st  and  2nd  group.  However,  by  its  giant 
colony,  its  vegetation  in  liquid  media  and  the  shape  of  its  spores,  it 
resembles  the  genera  Willia  and  Pichia. 

ZYGOSACCHAROMYCES  SALSUS.    Takahashi  and  Yukawa,  M.* 

This  species  was  discovered  in  samples  taken  from  all  the  fac- 
tories at  Tatsuna.  The  young  cells  from  the  surface  culture  on 
"Koji  "-extract  agar  are  mostly  round  (4-8  /-i)  and  rarely  oval.  The 
contents  are  homogeneous  and  sometimes  exhibit  vacuoles. 

On  streak  culture  at  27°  C.,  the  growth  shows  a  grayish  white, 
feeble,  finely  folded  covering.  On  glucose-sake-agar  after  10  days 
at  25°  C.  it  forms  a  grayish  yellow,  folded,  elevated  covering  with 
streamy  margin.  On  "Koji  "-extract  culture  at  23°  C.,  it  forms  a  few 
parts  of  yeast  ring  without  clouding  the  fluid  after  three  days.  The 
ring  gradually  grows  and  increases  its  thickness.  After  three  weeks 
a  thin  film  covers  the  surface.  The  culture  medium  which  was  kept 
for  three  months  was  strikingly  decolorized. 

Wort  culture  is  similar  to  the  former  culture,  but  this  yeast 
forms  a  grayish  white,  folded,  thick  film  on  "Shoju"  or  "Koji" 
extract  which  contains  a  quantity  of  NaCl.  This  yeast  is  easily  dis- 
tinguished from  Zygosaccharomyces  japonicus  by  this  characteristic 
point. 

This  species  ferments  dextrose,  levulose  and  maltose,  but  does 
not  ferment  galactose,  lactose,  saccharose,  raffmose,  a-methyl-glu- 
coside. 

In  formation  and  germination  of  spores  this  yeast  is  similar  to 
Zygosaccharomyces  japonicus,  but  the  time  required  for  sporulation 
of  this  yeast  is  longer  than  that  of  the  former  species. 

This  yeast  forms  a  thick  film  in  some  nutrient  fluids   which  con- 
tain a  quantity  of  NaCl,  but  not  in  the  absence  of  NaCl.     More- 

1  Takahashi,  and  Yukawa,  M.  Original  communications,  Eighth  Internatl. 
Congress  of  Applied  Chemistry,  y.  XIV,  1912,  p.  167. 


YEAST   F  219 

over,  this  yeast  is  easily  distinguished  from  the  former  species  by  the 
cell  forms,  and  the  time  limit  of  sporulation. 

Torida  soja  (G.  and  H.  Nishimura)  seem  to  be  identical  with  this 
species.  From  above  differentiation  we  gave  it  the  name  of  Zyg3- 
saccharomyces  salsus. 

ASPOROGENIC  SPECIES  OF  ZYGOSACCHAROMYCES 

Takahashi  and  M.  Yukawa  l 

On  the  mycological  relations  this  yeast  is  closely  similar  to  Zygo- 
saccharomyces  soja.  It  forms  a  well-defined  yeast  ring  in  "Shoju" 
and  "  Koji "  extract,  but  the  sporulated  cells  have  never  occurred 
in  any  yeast  ring  in  spite  of  the  presence  of  a  number  of  dumb-bell- 
shaped  cells. 

According  to  this,  this  yeast  seems  to  be  a  variety  of  Zygosac- 
charomyces  soja  which  has  lost  .the  capacity  of  forming  spores.  Sub- 
sequently we  have  continued  to  cultivate  this  yeast  in  various  nutrient 
media  for  restoring  the  power  of  sporogenation.  Whether  this 
yeast  has  lost  the  faculty  of  producing  spores,  temporarily  or  per- 
manently, has  not  been  determined. 

YEAST  F.     Pearce  and  Barker 

This  yeast  which  belongs  to  the  genus  Zygosaccharomyces  was 
discovered  by  Pearce  and  Barker2  in  1908  in  cider.  It  possesses  oval 
cells  (6.8  by  3.4  /*).  The 
maximum  temperature  for 
budding  is  situated  between 
30°  and  32.5°  C.  Sporula- 
tion  is  easily  accomplished 
on  porous  porcelain,  on 
potato  or  on  wort  gelatin. 
The  asc  which  results  is  com-  A-  B 

posed  of  two  enlarged  parts   Fig.    89. —Yeasts    F.     A,    Vegetative    Cells; 

.,    ,    ,  i        /T7-  B>  Ascs  (after  Pearce  and  Barker), 

united   by    a    canal.      (Fig. 

89,  B.)  The  ascospores  normally  to  the  number  of  four  are  situated 
two  in  each  enlarged  portion  of  the  asc.  Under  exceptional  circum- 
stances we  might  find  one  ascospore  in  one  enlargement  and  three 
in  the  other.  Germination  is  accomplished  by  a  swelling  of  the  as- 
cospore and  a  rupture  of  the  wall  of  the  asc;  this  is  followed  by  normal 

1  Takahashi,    and  Yukawa,    M.       Original  communications  eighth  internal! . 
Congress  of  Applied  Chemistry,  v.  XIV,  1912,  p.  167. 

2  Pearce,  B.  and  Barker,  P.    The  yeast  flora  of  bottled  ciders.    The  Journal 
of  Agricultural  Science,  3,  1908. 


220 


FAMILY  OF  SACCHAROMYCETACEAE 


budding.  Colonies  on  gelatin  plates  are  spherical  with  a  solid  appear- 
ance. In  streak  culture,  the  growth  is  slightly  humid  and  milky. 
This  yeast  ferments  levulose,  dextrose,  and  saccharose. 

YEAST  G.      Pearce  and  Barker 

This  species,  found  in  the  same  environment  as  the  former  yeast, 
generally  has  oval  cells.     The  maximum  temperature  for  budding  is 
around  32.5°  C.     (Fig.  90.)    Sporulation  has  been  obtained  on  porous 
porcelain;    it  is  preceded  by  a  process  which  seems  to 
be  intermediary  between  iso-  and  heterogamy.     The 
ascospores  are  formed  in  one  of  the  enlarged  portions 
of  the  asc.      (Fig.  23.)      Germination  is  accomplished 
as  in  Yeast  F.     This  species  develops  rather  rapidly 
on  beer  wort  and  in  all  sugar  solutions  with  a  scum, 
wnicn  tne  ce^s  possess  the  same  shape  as  those 
(after  Pearce  and  in  the  deposit.     Colonies  on  beer-wort  gelatin  are 
dry,    spherical    and     shriveled.       In     streaks,    the 
growth  is  slightly  bunched.     This  yeast  produces  no  fermentation. 


ZYGOSACCHAROMYCES    BISPORUS.     Anderson  * 

Morphology.     In  young  liquid  cultures  the  cells  are  oval  or  ovate ; 
in  old  cultures  they  assume  various  forms  with  numerous  conjugating, 
but  usually  no  sporulating  cells.      Elongated  cells 
are  common,  but  there  is  no  mycelial  formation. 
Budding  occurs  from  end  or  side.     The  size  is 
4  X  6.5  microns.      Spore    formation   occurs    on 
carrot  slants  at   room   temperature.      Conjuga- 
tion is  most  common  previous  to  spore  forma- 
tion, but  parthenogenesis  is  not  rare.    There  are 
2-4  ascospores,  most  commonly  2. 

Cultural  Characters.  On  glucose  agar  the 
growth  is  spreading,  dull,  flat,  and  white;  later 
it  becomes  brownish  with  small,  scattered,  wart- 
like  prominences  and  more  glistening  surface.  «•  Ag^rpicsianctfls  6ffroj£p££ 
There  is  a  filiform  growth  in  gelatin  stab  and  T  Ascofpore11  De^elop^fen^ 
liquefication  in  beer-wort  gelatin  in  3  weeks.  r^sSore^kiSEpmllt  ri 
Pellicle  is  present  on  beer  wort  and  some  sugar  (aftirgAnde?soS°njugation 
mediums. 

Physiologic   Properties.      It  does   not   ferment   glucose,    sucrose, 
levulose,  maltose,  galactose,  or  raffinose.     No  decided  change  in  acidity 


Anderson. 


.  —  Zygosac- 
bisporus, 


Anderson,  H.  W.     Yeast-like  fungi  of   the  human  intestinal  tract. 
Infectious  Diseases,  21  (1917)  341-386. 


Jour. 


DEBAROMYCES   GLOBOSUS  221 

occurs  in  these  mediums.      There  is  no  change  in  litmus  milk.      The 
culture  was  isolated  from  human  feces. 

Genus  III.     Debaromyces.     Klocker 

Ascs  derived  by  copulation.  Ascospores  in  a  single  membrane, 
the  surface  of  which  is  covered  with  little  elevations 

DEBAROMYCES  GLOBOSUS.    Klockei 

This  species  was  discovered  in  1909  by  Klocker  l  in  samples  of 
soil  from  the  island  of  Saint  Thomas.  On  beer  wort  at  25°  C.,  the 
cells  are  spherical  (4.5  to  5  microns  in  diameter).  The  limits  of  tem- 
perature for  budding  on  beer  wort  are:  maximum,  41.5  to  43;  mini- 
mum, 5  to  8°  C.  The  ascs  develop  abundantly  on  plaster  blocks  at 
32°  C.  The  limits  of  temperature  for  the  formation  of  ascospores  are: 
maximum,  34  to  36°  C.,  minimum,  14°  C. 

From  the  researches  of  Guilliermond,2  it  is  evident  that  the  ascs 
result  from  a  copulation.  In  about  25  per  cent  of  the  cases,  this  copu- 
lation is  by  isogamy  between 
two  cells  which  are  more  or  less  <£&*& 
closely  situated.  (Fig.  91.)  In 
all  of  the  other  cases  copulation 
occurs  between  an  adult  cell 
and  a  little  bud  which  was 
formed  by  this  cell,  but  which 
remained  attached  to  it  (Fig. 
28  c  and  91).  It  is  then  hetero- 
gamic.  The  yeast  may  then  be 
considered  as  a  form  in  which 

heterogamy  is  in  the  process  of    FiS-  91.  —  Copulation  and  Formation  of  the 
...        .      ...  „  Ascs  m  Debaromyces  globosus. 

installing  itself.     .Finally,  par- 
thenogenesis is  very  frequent,  more  than  in  the  Zygosaccharomyces. 

The  number  of  ascospores  varies  from  one  to  two  in  each  asc,  one 
being  more  frequent.  The  ascospores  are  globular  (2  to  3.5  /z).  Their 
surfaces  present  small  elevations.  In  the  center,  a  small  globule  of 
fat  is  found. 

1  Klocker,  A.    Deux  nouvelles  genres  de  la  famille  des  Saccharomyces.    Comp. 
Rend,  des  trav.  du  lab.  de  Oarlsberg,  8,  1909. 

2  Guilliermond,  A.     Sur  un  curieux  exemple  de  parthenogenese  observe  dans 
une  levure.    Comp.  Rend.  Soc.  Biol.  69,  1910.    Quelques  remarques  sur  la  sexua- 
lite  des  levures.    Annales  mycologici,  8,  1910;   Sur  la  reproduction  de  Debaromyces 
globosus  et  sur  quelques  phenomenones  de  retrogradation  de  la  sexualite  observes 
chez  les  levures.    Comp.  Rend.  Soc.  Biol.  151,  1911. 


222 


FAMILY   OF   SACCHAROMYCETACEAE 


During  germination  the  ascospores  swell  up  and  the  warts  on  their 
surfaces  disappear.  Germination  is  accomplished  by  ordinary  bud- 
ding. (Fig.  40.)  In  wort  cultures  no  scum  is  formed  but  often  a 
rudimentary  ring  may  be  observed.  Giant  colonies  on  gelatin,  at 
the  end  of  a  month,  have  a  grayish  white  color  resembling  wax.  The 
border  is  almost  entire;  the  center  is  slightly  raised  and  of  a  white 
color.  This  species  ferments  dextrose  and  levulose  actively,  raffinose 
moderately  and  inuline  with  difficulty.  It  has  no  action  on  maltose 
or  lactose.  It  inverts  saccharose  rapidly  and  ferments  it.  In  wort 
at  25°  C.  D.  globosus  produces  a  rather  rapid  fermentation.  After 
five  days,  it  forms  1.25  per  cent  of  alcohol  by  volume,  and  after  8 
days  1.30  per  cent. 

DEBAROMYCES  TYROCOLA.     Konokotin 

This  yeast  was  isolated  from  Dutch 
cheese  prepared  in  Russia.  It  is  a 
yeast  in  which  the  copulation  tends  to 
become  heterogamic.  The  copulation 
may  be  either  iso-  or  heterogamic  but 
the  latter  seems  to  be  most  frequent. 
It  is  accomplished  as  in  the  preced- 
ing species.  The  ascs  usually  contain 
a  single  ascospore  which  germinates 
by  budding.  This  species  ferments 
Fig.  91-A.  —  Debaromyces  tyrocola  dextrose,  levulose,  galactose,  saccha- 
(after  Konokotin).  rose  ancj  lactose. 


Genus  IV.     Nadsonia 

Ascs  preceded  by  a  heterogamic  copulation  between  a  mother  cell 
(macrogamete)  and  a  bud  from  it  (microgamete).  The  macrogam- 
ete  forms  a  bud  which  changes  into  an  asc  provided  with  one  asco- 
spore having  a  verrucose  wall. 


NADSONIA  FULVESCENS.    Sydon 
Syn.    GUILLIERMONDIA  FULVESCENS  (Nadson  and  Konokotin)  l 

This  yeast  was  found  along  with  Endomyces  magnusii.  It  bears 
some  resemblance  to  Debaromyces  globosus  but  differs  from  it  in  its 
sporulation  and  Nadson  and  Konokotin  have  created  a  new  genus  for  it. 

It  is  a  yeast  with  oval  cells,  elliptical  or  shaped  like  a  lemon. 

1  Nadson,  G.  A.  and  Konokotin,  A.  G.  Guilliermondia,  eine  neue  Hefengattung 
nait  heterogamer  Kopulation.  Cent.  Bakt.  34  (1912)  241-242. 


NADSONIA  ELONGATA 


223 


son  and  Konokotine)  . 

1-3,  Germination  of  Spores;   4,  Ascs;   5,  Cycle. 


The  asc  is  derived  from  a  heterogamic  copulation  of  two  cells.     The 

female  cell  or  macrogamete  is  an  adult  cell  and  the  male  cell  or 

microgamete  is  a  small  bud  formed  by  the  macrogamete.     Both  cells 

or  gametes  become  united  by  means  of  a  copulation  canal.    The  con- 

tents of  the  microgamete  enter  the  macrogamete  from  which  an  egg 

results.,    This  then  changes  into 

an    asc.     The   asc   contains    a 

single  ascospore.     The  spore  is 

spherical  with  a  large  globule 

of  fat  in  its  center.     Its  mem- 

brane is  rough  with  little  ele- 

vations and  has  a  reddish  brown 

color.     On  account  of  this  color, 

it  is  easy  to  recognize  macro 

scopically  a  culture  which  has 

sporulated.  During  germination 

the  spore  swells  up  and  breaks  Fig.  91-B.  —  Nadsonia  fulvescens  (after  Nad- 

open   the   wall.      Liberated,    it 

,          !  ,  .  ', 

develops  a  germinating  tube 
and  produces  a  vegetative  cell.  This  yeast  has  both  sporogenic 
and  asporogenic  types.  The  asporogenic  type  develops  into  colonies 
(giant)  which  are  distinguished  by  their  white  color  from  those  of 
the  sporogenic  which  are  reddish  brown.  This  species  ferments 
dextrose,  galactose,  levulose  and  saccharose  slowly. 

NADSONIA  ELONGATA.     Konokotin 

This  yeast  possesses  vegetative  cells 
which  are  oval  or  elongated.  Copulation 
is  accomplished  as  in  Nadsonia  fulvescens. 
The  asc  results  from  an  egg  containing 
a  single  spore.  These  ascospores  have  a 
very  verrucose  membrane  and  germinate 
by  ordinary  budding.  The  giant  colonies 
on  peptone  gelatin  with  5  per  cent  of 
glucose  are  rosette-shaped.  They  have 
brown  centers  and  white  peripheries. 
This  species  ferments  dextrose  and  levu- 
lose, but  has  no  action  on  other  sugars. 


Fig.  91-C.  —  Nadsonia  elongata 
(after  Konokotine). 


Genus  V.     Schwanniomyces.     Klocker 

Ascs  derived  from  cells  which  have  preserved  a  trace  of  sexual 
attraction.     Ascospore  provided  with  a  single  membrane  on  the  sur- 


224  FAMILY  OF  SACCHAROMYCETACEAE 

face  of  which  are  small  elevations,  and  also  provided  with  a  project- 
ing collar.  For  germination,  one  of  the  halves  of  the  ascospore  swells 
and  it  is  thus  that  budding  is  accomplished. 

SCHWANNIOMYCES   OCCIDENTALS.     Klocker 

This  species  was  found  by  Klocker  l  in  the  same  environment  as 
Debaromyces  globosus.  It  has  elliptical  or  spherical  cells  (5  to  10  JJL), 
but  some  cells  may  appear  rarely  as  elongated  sausages.  After  a 
month's  sojourn  at  room  temperature  on  beer  wort 
C  gelatin,  the  giant  colonies  appear  well  developed 

(o/^  with    a  grayish    white    appearance.     They   resemble 

wax  and  have  a  glistening  appearance. 

Sporulation    is    easily    accomplished    on    plaster 
blocks.     The    limits    of    temperature    for    ascospore 
formation  on  plaster  blocks  are:    minimum,  10  to  13° 
F-     92  _  Schw    C.;  maximum,  34°  to  36°  C.    The  ascs  are  always 
accident  alis   provided    with    a    projection  which   gives   them   the 
(after  Klocker).    appearance    of   a    retort  by  means   of  which,  during 

sporulation,  they  unite  two  by  two.      (Fig.  93.) 

Guilliermond  2  has  shown    that    these    formations    should    be  re- 
garded as  traces  of  an  ancestral  copulation.     The  cells  destined  to 
form   ascs   retain  a    little   of 
their    sexual    attraction    and       K     CT  ^0 


0  cO 


attempt  to  fuse.     Finally,  on          y  XD  /<T 
account  of  insufficient  sexual      "ft     C\ 
attraction,    the  cells  are  not         (g)  J>  04    <r5y~> 
able  to  establish  an  anasto-  ^AJV 

mosis  and  develop  partheno- 
genetically.  The  ascs  form 
usually  a  single  ascospore 
rarely  two.  The  ascospore  has 

the  shape  of   a    slightly  flat-    ^8-    93.  —  Formation  of   the  Asc   in  Schw. 

&      "  occidentahs. 

tened  bowl.    It  is  surrounded 

by  a  projecting  collar  which  divides  it  into  two  unequal  parts.  The 
surface  is  rendered  rugose  by  many  little  elevations.  In  the  center 
is  a  globule  of  fat. 

Germination  commences  by  a  swelling  of  the  ascospore  which  lo- 
calizes itself  to  the  smallest  half;  this  loses  its  elevations.  Finally 
all  of  the  elevations  disappear  (Fig.  41). 

1  Klocker,  A.    Deux  nouvelles  genres  de  la  famille  des  Saccharomyces.    Comp. 
Rend,  des  trav.  du  lab.  de  Carlsberg,  8,  1909. 

2  Guilliermond,  A.     Sur  un  curieux  exemple  de  parthenogenese  observe  dans 
une  levure.    Comp.  Rend.  Soc.  Biol.  69,  1910;   Quelques  remarques  sur  la  sexualite 
des  levures.     Annales  mycologici,  8,  1910. 


YEASTS  E   AND   F  225 

In  old  cultures  on  wort,  this  yeast  forms  a  viscous  scum  more 
or  less  developed.  Often  the  formation  of  a  very  thin  ring  may  be 
noticed. 

After  a  month's  sojourn  at  room  temperature,  giant  colonies  on 
wort  gelatin  appear  well  developed  with  a  grayish  color.  They  re- 
semble wax  and  possess  a  glistening  appearance.  The  edge  is  slightly 
notched. 

This  species  ferments  dextrose,  levulose,  and  raffinose,  sometimes 
inuline,  but  has  no  action  on  lactose  or  maltose.  It  inverts  sucrose 
more  or  less  actively. 

Genus  VI.     Torulaspora.     Lindner 

Cells  round,  spherical,  small,  provided  with  a  large  globule  of  fat 
and  resembling  Torula.  These  characteristics,  as  remarked  by  Klocker; 
are  insufficient  to  characterize  the  genus.  However  the  trace  of 
copulation  which  is  present  in  the  Torulaspora,  recently  pointed  out 
by  L.  Rose,  added  to  the  characters  described  by  Lindner,  seem  suf- 
ficient to  differentiate  this  genus. 

TORULASPORA  DELBRUCKII.    Lindner 

This  species  was  discovered  by  Lindner1  in  English  beer.  (Fig.  94.) 
The  ascospores  are  to  the  number  of  3  to  5  in  each  asc.  According  to 
Rose,  the  ascs  possess  spurs  analogous  to 

those  which  have  been  observed  in  the  C^tiT^     ^     O/O> 

Schwanniomyces  which  may  be  regarded       Ow^/rv^feA     (^T 
as  traces  of  copulation.    This  yeast  is       f^f^    r  ^-»  *  ^/ 
able  to  ferment  dextrose,  levulose,  d-man- 
nose  and  d-galactose. 


YEASTS    E    AND    F.     Rose 

®$\ 


This  species  was  isolated  from  the 
mucous  secretions  of  oak  trees  by  Rose2 
in  1910.  Both  present  the  characteristics 
of  the  genus  Torulaspora  and  seem  to  be  identical.  They  possess 
round  cells  (3.5  to  4.5  ju  in  diameter)  and  grow  on  beer  wort  quite 
well,  but  do  not  produce  fermentation. 

The   ascs   develop   at   the    end   of    three    days    on    Gorodkowa's 
gelatin  and  plaster  blocks  at  25°  C.     They  show  attempts  at  copula- 

1  Lindner,    P.      Mikroskopische    Betriebskontrolle    in    den    Garungswerben. 
Paul  Parey,  edit.  Berlin,  6th  edition,  1909. 

2  Rose,   L.     Beitrage   zur   Kenntniss  der  Organismen  in  Eichenschleimfluss. 
Inaugural  dissertation,  University  of  Berlin,  June  25,  1910. 


226  FAMILY  OF  SACCHAROMYCETACEAE 

tion  and,  like  the  Schwanniomyces,  are  supplied  with  a  projection  (Fig. 
30).  Often  one  may  see  a  union  of  two  of  these  projections  from 
closely  situated  cells  but  no  true  union  takes  place  on  account  of  the 
persistence  of  a  wall,  and  each  forms  a  parthenogenetic  asc. 

The  giant  colonies  are  flat  with  small  verrucose  elevations.  This 
yeast  ferments  dextrose,  levulose,  d-mannose,  and  saccharose  and 
sometimes  raffinose,  trehalose,  and  inuline. 

Rose  has  observed  traces  of  copulation  quite  similar  in  a  yeast  iso- 
lated by  Lindner  from  the  secretions  of  a  tree  in  the  Berlin  bo- 
tanical garden  and  described  in  his  atlas  as  Torula  sporogene  related  to 
Torulaspora  Delbruckii. 

SACCHAROMYCES  LACTIS  7.    Dombrowski 

This  yeast  isolated  by  Collau  from  sour  cream  butter  has  been  de- 
scribed by  Dombrowski.1  It  possesses  no  characteristics  of  the  genus 
Torulaspora.  However,  as  the  ascs  result  from  cells  which  have  pre- 
served a  trace  of  copulation  or  sexual  attrac- 
tion, it  should  probably  be  classed  along  with 
the  genera  Schwanniomyces  and  Torulaspora, 
preserving  the  provisional  name  of  Saccharo- 
myces  lactis  (gamma).  Perhaps  it  will  be  pos- 
sible to  create  a  new  genus  for  it  when  it  is 
better  known.  The  cells  are  oval,  sometimes 
spherical,  and,  on  beer  wort,  are  5  to  6.5  ju  in 

Cells     length  and  5  /*  in  breadth. 

destined  to  Form  ASCS;   3,         Sporulation  is  easily  accomplished  on  plaster 

Ascs  (after  Dombrowski).  .  -  .      .  ,     .         . 

blocks,  in  old  cultures  on  gelatin,  and  in  the 

moist  chamber.  They  appear  in  from  72  to  96  hours  on  plaster  blocks 
at  25°  C.  The  cells  destined  to  sporulate  show. the  presence  of  a  pro- 
jection more  or  less  elongated  which  seems  to  represent  a  trace  of 
ancestral  copulation  (Fig.  95,  2).  They  contain  large  fat  globules. 
The  ascs  contain  one  or  two  ascospores,  rarely  three.  (Fig.  95,  3.) 
The  ascospores  are  shiny  and  contain  a  drop  of  fat  in  the  center. 
Germination  is  accomplished  by  budding,  during  which  the  fat  dis- 
appears. 

In  cultures  on  nutrient  gelatin  in  plates,  the  colonies  are  round 
or  shaped  like  a  torpedo  in  which  the  edge  enlarges  in  old  cultures. 
In  gelatin  stabs  the  development  is  along  the  line  of  inoculation  and 
becomes  less  and  less  as  it  progresses  into  the  tube  away  from  the 
surface.  Giant  colonies  have  a  raised  center  and  a  border  composed 
of  concentric  rings  with  slender  rays. 

1  Dombrowski,  W.  Die  Hefen  in  Milch  und  Milchprodukten.  Cent.  Bakt. 
28,  1910. 


SACCHAROMYCODES   LUDWIGII  227 

In  wort  at  the  temperature  of  the  laboratory,  Saccharomyces  lac- 
tis'y  forms  a  ring  and  scum  in  which  the  cells  possess  the  same  charac- 
teristics as  those  in  the  sediment.  This  yeast  acts  like  a  top  yeast. 
Fermentation  is  active  at  first  but  ceases  quite  rapidly.  The  wort  is 
strongly  discolored.  No  aroma  seems  to  be  formed.  At  the  end  of 
five  months  and  a  half,  5.4  per  cent  of  alcohol  is  formed.  In  milk, 
this  yeast  produces  no  fermentation  but  provokes  a  strong  peptoni- 
zation  of  the  casein.  It  ferments  saccharose,  dextrose,  d-galactose, 
but  has  no  action  on  maltose  or  lactose. 


THIRD   GROUP 

Yeasts  multiplying  by  budding,  in  which  the  ascs  always  form 
by  parthenogenesis,  all  traces  of  sexuality  having  disappeared.  Some- 
times there  is  the  formation  of  a  rudimentary  mycelium.  In  sugar 
solutions  a  deposit  is  formed  and,  very  much  more  slowly,  a  scum, 
in  which  the  vegetation  is  shiny  without  occluded  bubbles  of  air.  The 
ascospores  are  smooth,  round  or  oval,  with  one  or  two  membranes, 
germinating  by  budding  or  under  exceptional  conditions  by  a  process 
intermediate  between  budding  and  partition.  Germination  of  the 
ascospores  is  sometimes  preceded  by  a  fusion  of  these  latter  two  by 
two  (parthenogamy).  The  greater  number  of  the  species  in  this  group 
produce  the  alcoholic  fermentation. 

Genus  VII.     Saccharomy codes.     Hansen 

Cells  dividing  by  a  process  intermediary  between  budding  1  and 
partition.  Often  there  are  rudimentary  myceliums  with  very  dis- 
tinct transverse  walls.  Ascopores  fuse  (parthenogamy)  two  by  two  at 
the  moment  of  germination  and  develop  in  a  single  direction  by  a 
process  intermediary  between  budding  and  partition. 

SACCHAROMYCODES  LUDWIGIL    Hansen 
Syn.    SACCHAROMYCES  LUDWIGII.     Hansen 

This  species  was  discovered  by  Ludwig  2  in  the  mucous  secretions 
of  the  oak  in  which  he  found  associated  a  Leuconostoc  and  Endomyces 
Magnusii.  Ludwig  first  regarded  it  as  a  form  of  Endomyces  mag- 

1  By  its  manner  of  multiplication,  the  genus  Saccharomycodes  is  then  inter- 
mediate between  the  Schizosaccharomyces  and  the  budding  yeasts.     It  may  also 
be  possible  to  separate  other  yeasts  into  a  separate  group. 

2  Ludwig,   F.     Ueber  der  Alkoholgarung  und  Schleimfluss  lebender  Baume. 
Bericht.  der  Deutsch.  Bot.  Gesells.  4,  1886. 


228  FAMILY  OF  SACCHAROMYCETACEAE 

nusii.    Hansen  l    later  isolated  Endomyces  magnusii.      Rose  has  found 
this  yeast  since  then  under  the  same  condition. 

Saccharomyces  Ludwigii  possesses  variable  shape  and  dimensions; 
some  cells  are  elliptical,  others  are  elongated,  tubular  or  with  the  shape 
of  a  lemon.  (Fig.  96.)  The  cells  multiply  by  a  process  intermediate 
between  budding  and  transverse  partition.  They  form  generally  at 
both  extremities,  rarely  laterally,  a  projection,  a  sort  of  a  bud  which, 
when  it  has  attained  a  certain  size,  separates  itself  from  the  mother 
cell  by  a  thin  wall  accompanied  by  a  slight  tightening  of  the  neck. 
The  temperature  limits  for  budding  in  beer  wort  are:  minimum, 
1-3°  C.;  maximum,  37-38°  C. 

In  old  cultures  especially  on  gelatin,  Saccharomyces  Ludwigii  shows 
a  manifest  tendency  to  produce  well  developed  rudimentary  myceliums 
which  resemble  a  true  mycelium.     These  forma- 
tions are  made  up  of  a  series  of  budding  ramify- 
ing filaments.    The  cross  walls  are  very  marked 
but  almost  always  accompanied  by  a  slight  con- 
striction and  the  units  easily  separate.     Each  of 
the  cells  in  the  mycelium   is  able    to    bud    and 
form  ascospores.     Long  branching  units  with  walls 
96.  —  Saccharo-   may  ^e  seen  m  the  mycelium.     (Fig.  5.)     By  the 
mycodes  Ludwigii  .    presence  of  these  mycelial  formations,  Sacch.  Lud- 
(after  Lindner).          w^  seems  to  offer  an  intermediate  step  between 

the  Endomyces  and  the  yeasts. 

Ascospores  form  easily  in  water  solutions  of  sugar,  on  wort  gela- 
tin in  yeast  water,  on  slices  of  carrot  and  even  in  liquid  wort.  They 
develop  equally  in  numbers  on  plaster  blocks. 

According  to  Nielsen2  the  maximum  temperature  for  sporulation 
on  plaster  blocks  is  32°  to  32.5°;  the  minimum  is  between  3°  and  6° 
and  the  optimum  between  30°  and  31°  C. 

The  ascs  may  contain  from  two  to  four  ascospores,  rarely  more, 
but  almost  always  there  are  four.  These  are  round  and  about  3  or 
4  /z  in  diameter.  Germination  is  accomplished  in  a  special  manner 
which  has  been  described  at  the  beginning  of  the  book  and  which  will 
not  be  repeated  here.  It  is  generally  preceded  by  a  sexual  process 
which  Guilliermond  3  has  described  and  which  is  comparable  to  par- 
thenogamy.  (Figs.  25  and  26.)  After  the  ascospores  have  swelled  they 

1  Hansen,  E.  C.     Ueber  die  im  Schleimfliisse  lebender  Baume  beobachteten 
Mikroorganismen.     Cent.  Bakt.  5,  1889. 

2  Nielsen,  J.  C.     Sur  le  dev.  des  spores  des  S.  membranaefaciens,  anomolous 
et  Ludwigii.     Comp.  Rend,  des  trav.  du  lab.  de  Carlsberg,  3,  1891. 

3  Guilliermond,  A.     Recherches  sur  la  germination  des  spores  et  sur  la  con- 
jugaison  dans  les  levures.     Rev.  gen.  de  Bot.  17,  1905. 


SACCHAROMYCES   BEHRENSIANUS  229 

unite  two  by  two.  This  fusion  almost  always  operates  between  two 
ascospores  from  the  same  asc,  exceptionally  between  two  spores  from 
closely  situated  ascs.  A  canal  for  copulation  is  formed  through 
which  the  contents  fuse.  The  fusion  takes  place,  the  copulation  canal 
gives  birth  to  a  sort  of  germinating  tube  which  enlarges  and  takes  the 
shape  of  a  vegetative  cell ;  it  finally  cuts  itself  off  from  the  canal  by 
a  wall  accompanied  by  a  circular  construction.  (Fig.  36.)  The  cell 
thus  formed  separates  from  the  zygospore  which  continues  to  form 
new  cells  by  the  same  process.  The  fusion  of  the  ascospores  is  gen- 
erally not  absolute  and  quite  a  number  among  them  germinate  alone. 

We  have  observed,  as  has  been  stated,  a  species  of  Saccharomyces 
Ludwigii  from  Hansen's  laboratory  which,  having  remained  for  a  time 
at  laboratory  temperature,  had  completely  lost  its  sexuality.  The 
ascospores  always  developed  without  fusing. 

Hansen  has  shown  that  when  various  cells  are  cultivated  from  a 
colony  of  this  yeast,  a  sporogenic  and  an  asporogenic  race  may  be 
obtained. 

On  wort  gelatin,  Saccharomyces  Ludwigii  develops  in  the  shape  of 
vegetative  spots  in  which  the  color  varies  from  a  clear  gray  to  a  pale 
yellow.  In  beer  wort,  it  produces  at  the  end  of  about  a  month,  at 
room  temperature,  a  scum  with  elongated  colonies. 

It  yields  even  after  a  fermentation  of  long  duration,  only  1.2  per 
cent  of  alcohol  by  volume.  It  does  not  act  on  maltose.  On  the  other 
hand  in  glucose  about  10  per  cent  of  alcohol  is  produced.  It  inverts 
saccharose,  and  ferments  dextrose,  d-galactose,  d-mannose,  levulose, 
raffinose,  and  sometimes  very  slightly,  1-sorbose,  and  tagatose  (Lind- 
ner). It  has  no  action  on  lactose  or  maltose. 


SACCHAROMYCES  BEHRENSIANUS.    (Behrens) 

This  yeast,  discovered  by  Behrens  l  on  hops,  possesses  round  or 
oval  cells  which  divide  like  those  of  Saccharomyces  Ludwigii.  The 
optimum  temperature  for  sporulation  is  from  18  to  20°  C.;  at  this 
temperature,  the  ascospores  appear  in  about  22  hours.  The  asco- 
spores are  spherical  (4  to  4.5/>t  in  diameter)  and  are  to  the  number  of 
two  or  three  in  an  asc.  Their  germination  is  accomplished  as  in  Sac- 
charomyces Ludwigii.  This  yeast  produces  no  scum.  On  10  per 
cent  wort  gelatin,  the  giant  colonies  present  quite  a  characteristic 
appearance.  They  show  fine  concentric  rings  placed  around  a  cra- 
teriform  cavity  which  makes  up  the  center.  The  edge  of  the  colonies 
is  of  a  pure  white  and  the  middle  portions  of  a  yellow  color.  In 

1  Behrens,  J.  Studien  iiber  die  Konswevierung  und  Zusammensetzung  des 
Hopfens.  Woch.  f.  Brau.  13,  1896. 


230  FAMILY  OF  SACCHAROMYCETACEAF 

giant  colonies,  numerous  ascospores  are  noticed.  This  yeast  ferments 
dextrose,  levulose,  and  maltose  but  does  not  act  on  saccharose,  lac- 
tose, or  d-galactose. 

SACCHAROMYCES  COMESII.     Cavara 

This  species  was  described  in  1893  by  Cavara.1  It  grows  parasit- 
ically  and  saprophytically  in  the  panicles  and  stalks  of  millet.  Cavara 
described  ascs  enclosing  a  varied  number  of  ascospores.  These  asco- 
spores fuse  into  one  at  the  moment  of  their  germination.  Germina- 
tion is  accomplished  as  with  Saccharomyces  Ludwigii  by  the  forma- 
tion of  a  germinating  tube.  Guilliermond 2  has  shown  from  the 
illustrations  presented  by  Cavara,  that  this  species  is  probably  not 
yeast  but  probably  a  Dematium.  Cavara  regarded  it  as  a  yeast  on 
account  of  the  incorrect  interpretation  of  forms  in  its  development. 
Saccharomyces  comesii  is,  then,  not  a  yeast  and  should  not  be  included 
in  the  family  of  Saccharomycetes.  . 

Genus  VIII.     Saccharomycopsis  (Schionning) 
Ascospores  in  two  membranes  germinating  by  budding. 

SACCHAROMYCOPSIS  GUTTULATUS  (Robin)     Schionning 

Syn.     SACCHAROMYCES  GUTTULATUS.     Winter.     CRYPTOCOCCUS 

GUTTULATUS.      Robin 

This  species  was  discovered  by  Remarck  and  Robin  and  studied 
later  b^  Buscalioni,3  Casagrandi  and  Wilhelmi.4  It  seems  to  live  as  a 

true  parasite  in  the  intestinal  canal 
of  certain  animals  (birds,  reptiles 
and  mammals).  It  swarms  in  the 
intestinal  canal  of  the  rabbit,  less 
frequent  in  guinea  pigs,  and  ap- 
pears in  the  excrement  of  these 

animals.     Saccharomyces  guttulatus 
Fig.    97.  —  Saccharomycopsis    quttulatus  ,  n  -, 

(after  Buscalioni).  possesses  large  cells,  oval  or  more 

or  less  rectangular,  resembling  the 

oidia  of  Oidium  lactis.  The  cells  contain  a  large  amount  of  glycogen 
and  are  often  united  in  groups  at  their  ends.  (Fig.  97.)  Budding 

1  Cavara.     Sur  un  microorganisme  zymogene  de  la  Dourra.      Revue  mycolo- 
gique,  1893. 

2  Guilliermond,   A.     Observations  sur  la  germination  des  spores  du  S.  Lud- 
wigii.    Bull,  de  la  Societe  de  Mycologie,  19,  1903. 

3  Buscalioni,  L.      Saccharomyces  guttulatus.      Giornale   Malphigia,    10,    1896. 

4  Wilhelmi,  A.     Beitrage  zur  Kenntniss  des  Saccharomyces  guttulatus.     (Bus- 
calioni) Inaugural  dissertation.     Bern.  Cent.  Bakt.  4,  1898. 


SACCHAROMYCES  CEREVISIAE  231 

takes  place  at  both  ends  of  the  cells.  The  optimum  temperature  for 
budding  is  from  35°  to  37°  C.  A  scum  formation  has  not  been  ob- 
served. Sporulation  has  been  observed  in  the  excrements  of  rabbits. 
Ascospores  are  formed  to  the  number  of  one  to  four  in  each  asc.  They 
are  oval,  elongated  and,  according  to  Wilhelmi,  are  surrounded  with  a 
double  membrane,  an  exosporium  and  an  endosporium.  Wilhelmi  has 
been  able  to  cultivate  Saccharomyces  guttulatus  in  various  artificial 
media.  It  grew  especially  well  in  glycerol  gelatin  to  which  tartaric 
acid  and  dextrose  had  been  added.  It  inverts  saccharose  and  ferments 
dextrose.  Casagrandi  and  Buscalioni  have  noted  its  pathogenic  proper- 
ties on  subcutaneous  injections  into  guinea  pigs,  rats  and  rabbits. 

Genus  IX.     Saccharomyces.     Meyen 

Ascospores  in  a  single  membrane  germinating  by  budding.  Some- 
times a  mycelium  is  produced  with  transverse  walls. 

A.     First  Sub-group 

Yeasts  fermenting  saccharose,  dextrose  and  maltose  but  having  no 
action  on  lactose. 

SACCHAROMYCES   CEREVISIAE.     Hansen1 

Syn.     s.  CEREVISIAE  i,   Hansen.  —  s.   CEREVISIAE,  Meyen.  —  TORULA 
CEREVISIAE,  Turpin.  —  CRYPTOCOCCUS  FERMENTUM,  Kutzing. — 
HORMISCUM  CEREVISIAE,  Bail.  —  s.  CEREVISIAE,  Rees 

This  species  is  a  top  yeast  which    was    found    by    Hansen    in 
breweries  of  London  and  Edinburgh  and 
has  been  used  for  a   long   time  in  the 

making  of  beer.     Hansen    gave    it    the    ^O^xgf  ^         Q\ 
name  S.  cerevisiae  because  it  resembled    Virxi^'n^n     \J 
the    yeast    described    under    the    same 
name  by  Rees  and  Meyen.    Young  cells 
in  the  sediment  in  beer  wort  are  large  and 
either   round   or   oval.     Elongated  cells 
are    not    observed    under    these    condi-  Fig.   98.  —  Ascs  of  S.  cerevisiae 
tions.     (Fig.  2.)    The  temperature  limits 
for  budding  in  beer  wort  are:   minimum,  1-3°  C.;  maximum,  40°  C. 

1  Hansen,  E.  C.  Recherches  sur  la  morphologic  et  la  physiologic  des  alcoo- 
liques  ferments.  II,  Les  ascospores  chez  le  genre  Saccharomyces.  Ill,  Sur  la 
Torula  de  M.  Pasteur.  IV,  Maladies  provoques  dans  la  biere  par  les  ferments 
alcooliques.  Comp.  rend,  du  lab.  de  Carlsberg,  Vol.  1,  Book  2,  1883;  Vol.  2, 
Book  4;  Vol.  5,  Book  2,  1909. 


232 


FAMILY  OF  SACCHAROMYCETACEAE 


Fig.  98-A.  —  Chondri- 
ome  in  Saccharomyces 
cerevisiae. 

Fixation  in  a  mixture  of 
Potassium  Dichromate  and 
Formalin.  Stained  with 
Ferric  haematoxyline. 


Temperatures  for  the  formation  of  ascospores 
37.5°  C.  no  ascospores  are  formed 

36-37  first  appearances  in  about  29  hours 

25  " 
23 
20 
23 
27 
50 
65 
10  days 


35 
33.5 
30 
25 
23 
17.5 
16.5 

11-12     " 
9      no  development 


The  ascs  enclose  from  one  to  four  ascospores,  sometimes  five.  The 
ascospores  are  very  refractive  and  possess  a  very  distinct  wall.  (Fig. 
98.)  Their  size  varies  from  2.5  to  6  jit. 


Temperatures  of  Scum  Formation 
At  38°  C.  there  is  no  formation  of  scum. 

33-34  at  the  end  of    9-18  days,  very  slightly  developed 
26-28  "     "      "     "     7-11 
20-22   "     "      "     "     7-10 
13-15  "     "      "     "   15-30 
6_7     «    «       «     «     2-3  months    " 
5  no  formation  of  scum. 

The  cells  in  the  scum  have  the  following  micro- 
scopic characteristics.  At  20-34°  C.  the  cells  in  the 
scum  are  elongated  and  possess  an  odd  appearance. 
At  15  to  16°  C.,  most  of  the  cells  look  like  those 
which  were  present  when  the  inoculation  was  made. 
Some  of  the  cells  possess  irregular  shapes.  In  old 
scums  all  sorts  of  cells  are  visible.  Some  are  ex- 
tremely long,  having  the  appearance  of  a  mycelium. 
(Fig.  99.) 

Yeasts  like  S.  Cerevisiae 

Numerous  races  and  species  of  yeasts  are  known  _ 

under  the  name  of  S.  cerevisiae.    Their  systematic      visiae.       Mycelial 
position  is  slightly  unknown;   a  few  of  them  will  be 
mentioned  here.  Hansen) 


YEASTS  OF  SAAZ   AND   FROHBERG  233 

WILL'S  YEASTS 

Variety  2,  H.  Will.1  Large  round  or  oval  cells.  On  gelatin  the 
colonies  are  spherical  or  lenticular.  Ascospores  develop  easily  and 
abundantly.  The  temperature  limits  for  the  formation  of  ascospores 
on  plates  are  31°  and  11°  C.,  the  optimum  being  near  25°  or  26°  C. 
The  temperature  limits  for  scum  formation  on  beer  wort  are  from  28 
-31°  C.  to  8-11°  C.  This  variety  is  a  bottom  yeast  which  is  an  ac- 
tive fermenter. 

Variety  6,  H.  Will.  The  cells  of  this  variety  are  oval  and  some- 
times curled.  The  colonies  are  spherical  or  lenticular  on  gelatin. 
Ascospores  are  formed  easily  and  abundantly.  The  temperature 
limits  for  ascospore  formation  on  plaster  blocks  are  31°  and  11°  C.  The 
optimum  is  28°  C.  The  temperature  limits  for  scum  formation  are 
25-31°  and  7-10°.  This  species  is  a  moderate  fermenter  and  a  bottom 
yeast. 

Variety  7,  H.  Will.  The  cells 'are  round  with  giant  cells  mixed  in. 
At 'the  end  of  fermentation,  chains  may  be  seen  composed  of  small 
cells  in  the  budding  stage.  The  colonies  on  gelatin  are  at  first  ir- 
regular with  a  winding  embattled  edge.  Ascospores  are  formed  with 
difficulty.  The  temperature  limits  for  the  formation  of  ascospores 
on  plaster  blocks  are  30°  and  13°.  The  optimum  is  around  25  or  26°  C. 
The  limits  of  temperature  for  the  formation  of  scum  on  beer  wort 
are  20-28°  and  4-7°  C.  This  variety  is  a  bottom  yeast  with  feeble 
fermenting  ability. 

Variety  93,  H.  Will.  The  cells  are  round  or  oval  and  the  gelatin 
colonies  spherical  or  lenticular.  The  temperature  limits  for  the  for- 
mation of  ascospores  on  plaster  blocks  are  30°  and  10°  C.  The  opti- 
mum is  28°  C.  The  temperature  limits  for  scum  formation  on  beer 
wort  are  30-31°  and  4-7°  C.  The  scum  contains  numerous  durable 
cells.  This  variety  is  a  very  active  bottom  yeast. 


YEASTS  OF  SAAZ  AND  FROHBERG 

Although  insufficiently  known  from  the  standpoint  of  morphology, 
we  should  not  pass  these  yeasts  in  silence  for  they  play  an  impor- 
tant role  in  the  industrial  alcoholic  fermentations.  One  was  found  in 
a  Bohemian  brewery  (Saaz),  the  other  in  a  brewery  at  Frohberg 
having  been  isolated  by  Lindner.2  Since  that  time  they  have  been 

1  Will,  H.     Vergleichende    Untersuchungen    an  vier  untergarigen  Arten  von 
Bierhefe.     Zeitschr.  f.  d.  ges.  Brauw.  18,  1896. 

2  Lindner,    P.      Mikroskopische    Betriebskontrolle    in    den    Garungswerben. 
Paul  Parey,  Berlin,  6th  Edition,  1909. 


234  FAMILY  OF  SACCHAROMYCETACEAE 

studied  by  various  investigators  as  Delbriick,  Irmisch,  Lindner, 
Reinicke,  etc.  The  Saaz  yeast  is  a  bottom  yeast  by  attenuation  and 
ferments  dextrose,  d-mannose,  d-galactose,  levulose,  maltose,  saccha- 
rose, trehalose,  melibiose,  raffinose  and  a-methylglucoside. 

A  large  number  of  industrial  species  (brewery  and  distillery) 
related  to  these  species  have  been  described.  Some  produce  a  top 
fermentation  while  others  produce  a  bottom  fermentation.  Bau  has 
made  four  groups:  1,  Bottom  yeasts  of  the  Saaz  type;  2,  Top  yeasts 
of  the  Saaz  type;  3,  Bottom  yeasts  of  the  Frohberg  type;  4,  Top 
yeasts  of  the  Frohberg  type.  The  yeasts  of  the  first  two  groups 
are  characterized  by  the  fact  that  they  give  a  feeble  attenuation, 
those  of  the  second  two  groups  that  they  give  a  strong  attenuation. 

Different  industrial  yeasts  have  been  described  by  Van  Laer,  Jor- 
gensen,  Greg,  and  a  few  other  authors.  Among  the  best  known  may 
be  mentioned  variety  II  and  XII,  top  distillery  yeasts  which  have 
been  isolated  at  the  Berlin  Institute  of  Fermentology. 


SACCHAROMYCES  CARLSBERGENSIS.    Hansen 
Syn.     CARLSBERG  YEAST.     Hansen 

Described  for  a  long  time  by  Hansen  1  under  the  provisional  name 
of  Carlsberg  Yeast  I,  this  species  has  been  subjected  to  a  careful 

study  by  the  same  author  who  has  given  it 
^e  name  °f  Saccharomyces  Carlsbergensis. 
This  yeast  like  the  Saccharomyces  monacensis 
which  we  shall  describe  shortly,  has  been 

_.  used  for  a  long  time  in  Copenhagen  breweries. 

Fig.  100.  —  S.  carlsbergensts.  nA      ^ 

A    Wort    Culture    of    24  After  culturmg  for  24  hours  on  beer  wort 
Hours  at  25°  (after  Han-   at  25°  C.,  or  after  two  days  at  the  tempera- 
ture of  the  laboratory,  Saccharomyces  carls- 

bergensis  forms  a  doughy  sediment  made  up  of  elliptical  cells,  shaped 
like  an  egg  or  pear.  (Fig.  102.)  Cells  with  small  points  make  up  the 
characteristic  shape  of  this  variety.  The  temperature  limits  for  bud- 
ding on  beer  wort  are  33.5°  C.  and  0°  C. 

In  the  neighborhood  of  the  minimum  temperature,  this  yeast  forms 
mycelial  cells  in  chains  with  elliptical  cells  intermixed.  Giant  cells 
are  rare.  The  mycelial  cells  form  between  0°  and  9°  C.  (Fig.  101.) 
They  appear  after  2  or  3  months  at  0.5°  C.  At  1  or  2°  C.  they  are 
formed  in  a  month  and  a  half  and  a  little  more  quickly  at  3  or  4°  C. 

1  Hansen,  E.  C.  Recherches  sur  la  physiologic  et  morphologic  des  ferments 
alcooliques,  XIII.  Nouvelles  Etudes  sur  des  levures  de  brasserie  a  fermentation 
basse.  Comp.  Rend.,  lab.  de  Carlsberg,  7,  1908. 


SACCHAROMYCES   MONACENSIS 


235 


At  the  maximum  temperature,  the  cells  have  the  same  shape  as 
the  cells  used  in  the  inoculation  except  that  they  are  a  little  larger. 
On  the  other  hand  giant  cells  are  more  numerous. 
(Fig.  102.)  Ascospores  are  formed  but  rarely  and 
in  small  numbers.  It  is  not  possible  to  determine 
the  temperature  limits  for  their  development.  A 
feeble  formation  of  scum  is  obtained  in  Pasteur 
flasks  after  a  month  at  13°  to  15°  and  at  laboratory 
temperature.  At  the  end  of  two  months,  little 
islands  of  scum  have  formed  made  up  of  spherical 
and  elliptical  cells.  At  the  end  of  one  or  two  years, 
the  old  scum  covering  cultures  maintained  at  the 
temperature  of  the  laboratory  shows  the  same  Fig.  101. — S.  carls- 
characteristics.  The  cells  are  always  spherical  or 
ellipsoidal  and  the  chain  formation  is  very  rare. 

Giant  colonies  on  wort  gelatin  offer  the  form  of 
a  rosette.    More  often  they  present  in  the  center  a 

depression  which  is  surrounded  by  a  number  of 
concentric  rings.  The  edge  of  the  colony  is 
slightly  undulated.  Some  of  the  colonies  have  a 
glossy  surface  while  others  have  a  scaly  appear- 
ance. They  are  ordinarily  dry  and  of  a  chalky 

10    s       ,     appearance.    Liquefaction  of  gelatin  is  not  pro- 

bergensis.  Vegetation  duced  in  three  months. 

of  a  Culture  after          Cultures  on  plates  or  on  gelatin  produce  colo'nies 
two  to  three  months  *,     .         .     .  ,        *'       m  /--t 

in  Beer  Wort  at  32-  of  the  shape  and  size  of  pinheads.     The  superficial 

35°  C.  (according  to   colOnies  have  a  grayish  yellow  color  and  a  chalky 
appearance.     This  yeast  ferments  dextrose,  sac- 
charose, maltose  and  galactose,  but  not  lactose;  it  is  a  bottom  yeast 
and  an  active  fermenter. 


bergensis.  Vegeta- 
tion after  Two  or 
Three  Months  in 
Beer  Wort  at  0.5° 
C.  (after  Hansen). 


SACCHAROMYCES  MONACENSIS.    Hansen1 

This  species  described  for  a  long  time  under  the  provisional 
name  of  Carlsberg  Yeast  II  has  recently  been  restudied  by  Hansen 
and  given  the  definite  name  of  Saccharomyces  monacensis.  In  cul- 
tures on  beer  wort,  it  forms  at  the  end  of  24  hours  at  25°  C.  a  thin 
waxy  sediment  made  up  mostly  of  ellipsoidal  or  round  cells.  Giant 
cells  are  rather  frequent. 

The  temperature  limits  for  budding  in  beer  wort  are  33°  and  1°  C. 

1  Hansen,  E.  C.  Recherches  sur  la  morphologic  et  la  physiologic  des  fer- 
ments alcooliques.  XIII.  Nouvelles  etudes  sur  des  levures  de  brasserie  a  fer- 
mentation basse.  Comp.  Rend,  du  lab.  de  Carlsberg,  7,  1908. 


236 


FAMILY  OF  SACCHAROMYCETACEAE 


Fig.  103.  —  S.  monacensis. 

I.  Vegetation  of  a  Culture  on  Beer  Wort  after  2  to  3  Days  at 
32-33°C.  —  2.  Giant  Cells  Obtained  in  a  Culture  of  10  Days 
in  Yeast  Water  to  Which  Dextrose  Was  Added  at  9°  C.  — 
3.  Vegetation  after  16  Days  on  Beer  Wort  at  1-2°  C.  (after 
Hansen). 


At  the  maximum  temperature,  this  yeast  has  ellipsoidal  cells  predomi- 
nating with  a  few  in  the  form  of  chains.  The  cells  are  larger  and 
longer  (Fig.  103,  1);  at  the  minimum  temperature,  spherical  and 
ellipsoidal  cells  may  be  seen  at  the  end  of  18  days.  At  the  end  of  a 

month,   one  may  find 
^^ 


small  number  of  colonies 
composed  of  cells  in  a 
short  chain  with  a  few 
rare  giant  cells.  (Fig. 
103,  3.) 

In  yeast  water  to  which 
dextrose  has  been  added, 
one  may  notice  after  about 
12  days  at  9°  C.  a  feeble 
formation  of  a  mycelium. 
On  the  other  hand,  in 
this  medium  giant  cells  are  so  numerous  and  so  large  that  they  serve 
as  a  distinguishing  characteristic  between  this  species  and  the  preced- 
ing one.  (Fig.  103,  2.) 

At  13°  to  15°  C.,  one  may  see  at  the  end  of  about  a  month  in  a 
Pasteur  flask,  the  beginning  of  a  scum  with  the  form  of  floating  islands 
in  which  the  cells  are  spherical  or  ellipsoidal.  At  the  end  of  a  year, 
one  may  see  in  cultures,  maintained  at  labora- 
torjr  temperatures,  an  abundant  scum  formation 
which  generally  covers  about  all  of  the  surface. 
These  scums  are  again  composed  of  cells  which 
are  ellipsoidal  or  round,  chain  formation  being 
extremely  rare. 

Ascospore  formation  is  accomplished  more 
easily  in  this  species  than  in  the  preceding  one, 
without  being  abundant.     (Fig.  104.)    But  it  is  impossible  to  deter- 
mine the  temperature  limits  of  this  formation. 

Giant  colonies  on  wort  gelatin  have  much  the  appearance  of 
those  of  the  preceding  species.  They  take  the  shape  of  rosettes  with 
an  undulating  border.  The  colonies  have  a  depressed  center  which  is 
surrounded  by  a  more  elevated  ring  than  with  S.  carlsbergensis.  How- 
ever the  center  of  the  colony  consists  more  often  of  a  wart  and  the 
points  of  the  rosette  are  a  little  more  pronounced  than  in  S.  carls- 
bergensis. S.  monacensis  is  a  yeast  producing  a  typical  bottom  fermen- 
tation, fermenting  dextrose,  saccharose,  maltose  and  not  lactose. 


Fig.  104.  —  S.  monacen- 
sis, with  Ascs  (after 
Hansen) . 


SACCHAROMYCES   PASTORIANUS 
SACCHAROMYCES  PASTORIANUS.    Hansen l 


237 


This  is  a  species  which  is  often  encountered  in  the  air  in  the  vicin- 
ity of  breweries.  It  contributes  a  bitter,  disagreeable  taste  and  bad 
odor  to  the  beer.  Thus  it  contributes  to  the  diseases  of  beer  and  im- 
pedes the  clarification.  It  resembles  very  much  Saccharomyces  pas- 
torianus  described  by  Rees  and  Pasteur  but  is  not  capable  of  being 


Fig.  105.  —  Ascs  of  S. 
Pastorianus  (after 
Hansen) . 


Fig.  106.     S.  Pastorianus.     Vegetative  Cells  from 
Scum  at  13-15°C.  (after  Hansen). 


closely  identified  with  this  species.  It  forms  in  beer  wort  a  sediment  in 
which  the  cells  are  more  or  less  elongated,  mixed  with  large  and  small 
round  or  oval  cells.  (Fig.  3.) 

The  temperature  limits  for  budding  in  beer  wort  are:    minimum 
0.5°  C.,  maximum  34°  C. 

Temperatures  for  Ascospore  Formation 
At  31. 5°  C.  no  formation  of  ascospores. 

29.5-30.5°  first  appearance  of  rudimentary  ascospores  in  30  hours. 


29° 

27.5° 

23.5° 

18° 

15° 

10° 
8.5° 
7.0° 
3  -4° 
0.5° 


27 
24 
26 
35 
50 
89 

5  days 

6  " 
14     " 


no  formation  of  ascopores. 

1  Hansen,  E.  C.  Recherches  sur  la  physiologic  et  la  morphologie  des  ferments 
alcooliques.  II.  Les  ascospores  chez  le  genre  Saccharomyces.  III.  Sur  la 
Torula  de  M.  Pasteur.  IV.  Maladies  provoques  dans  la  biere  par  les  ferments 
alcooliques.  Comp.  Rend,  du  lab.  de  Carlsberg.  Vol.  1,  Book  2,  1883;  Vol.  2, 
Book  4,  1886;  Cent.  Bakt.  Vol.  5,  Book  2,  1909. 


238  FAMILY  OF  SACCHAROMYCETACEAE 

The  ascs  are  elongated  (Fig.  105)  and  possess  a  number  of  asco- 
spores  which  vary  from  1  to  4  and  may  attain  the  number  of  5  or  10. 
The  size  of  the  ascospore  is  quite  variable;  it  varies  between 
1.5  and  3.5  microns  and  goes  rarely  to  5.0  microns.  The  ascospores 
never  undergo  copulation,  which  distinguishes  S.  pastorianus  from 
S.  intermedium  and  S.  validus  (Marchaud). 

Temperatures  of  Scum  Formation 
At  34°  no  scum  formation. 

26-28°  at  the  end  of  from  7  to  10    days,  feebly  developed. 
20-22°   "     "•     "     "      "     8  to  15       "          "  " 

13-15°   "     "      "     "      "    15  to  30       " 
6-7°     "     "      "     "      "      1  to    2  months    " 
3-5°     "     "      "     "      "     5  to    6       "          "  " 

2-3°     no  formation  of  a  scum. 

The  microscopic  appearance  of  the  cells  in  the  scum  is  as  follows: 
At  20-28°  C.  the  cells  present  the  same  shape  as  in  the  deposit.  At 
13  to  15°  C.,  vigorous  colonies  are  seen  having  the  appearance  of  a 
mycelium  composed  of  ordinary  cells  elongated  and  in  the  form  of 
chains  (Fig.  106).  In  old  cultures  of  scums,  the  cells  are  smaller 
than  in  the  sediment.  One  finds  queer  cells  sometimes  almost  filiform. 
This  yeast  ferments  saccharose,  dextrose,  levulose  and  maltose. 


SACCHAROMYCES  INTERMEDIUS.     Hansen1 
Syn.  SACCHAROMYCES  PASTORIANUS  II.    Hansen.    Also  Rees 

This  yeast  produces  a  feeble  top  fermentation.  It  was  discovered 
by  Hansen  in  the  air  of  breweries  in  Copenhagen. 
It  does  not  seem  to 
cause  any  disease  in 
the  beer.  Hansen 
has  provisionally 
named  it  Saccharo- 

myces  pastorianus  II.   Fig.  107-A.  —  Germination  of  As- 
In    wort   it  forms   a       cospores  in  Saccharomyces  inter- 
-  medius. 

from    Sediment    in    sediment     of     elon-   i,  without  Copulation;' 2-9,  Af ter  Copu- 

gated  cells  in   chain 

formation.  (Fig.  107.)  One  finds  also  large  and  small  round  or  oval 
cells.  The  temperatures  of  budding  on  beer  wort  are:  minimum, 
0.5°  C.;  maximum  40°  C. 

1  Hansen,  E.  C.    See  references  for  Saccharomyces  pastorianus. 


j  *» 

ff 


i 


SACCHAROMYCES  INTERMEDIUS 


239 


Temperatures  for  the  Formation  of  Ascospores 
At  29°  C.  no  formation  of  ascospores. 

27-28°  appearance  of  first  rudiments  in  34  hours. 

25°  "  "     "  "         "  25 

000  tl                          tt           tt                               tt                     it  O7 
-1ITO                                    tt                         tl           It                             tt                    it  0£» 

1  CO  It  It          tt  tt 

11.5°  "  "     " 

fJO  It          tt     tt  tt        tl  rj 

tl          It     It  tt        It  -tfj 


tt 
ti 
it 
u      AO       (( 

U       n  U 


0.5°  no  formation  of  ascospores. 

The  ascs  are  often  elongated  and  include  a  variable  number  of 
ascospores  (1  to  7)  which  measure  2  to  5  microns  in  diameter.  (Fig. 
108.)  The  ascospores  undergo  a  copulation  at  the  moment  of  ger- 
mination in  about  half  of  the  cases. 


Temperatures  for  the  Formation  of  Scum 
At  34°  C.  no  formation  of  ascospores 

26-28°  at  the  end  of    7-10  days  feeble  development. 


8-15 
"  10-25     " 

"     1-2    months 

tt 


20-22° 
13-15°   " 

6-7°     " 

3-5°     " 

2-3°  no  formation  of  scum. 


Microscopic  appearance  of  cells  in  the  scum  is  as  follows:  At 
20-28°  C.  the  cells  of  the  scum  present 
almost  the  same  shape  as  those  in 
the  sediment.  One  may  see  queer 
shapes  in  the  forms  of  chains.  At  15 
to  3°  C.  oval  or  round  cells  predomi- 
nate. In  the  scums  from  old  cultures, 
the  cells  are  almost  all  smaller  than 
those  in  the  sediment.  In  yeast 
water  gelatin,  on  streaks  at  15°  C., 
this  yeast  gives,  at  the  end  of  about  Fig.  108. — Ascs  of  S.  intermedius 
16  days,  a  vegetation  with  relatively 

even  edge  which  differs  from  Saccharomyces  validus.     It  ferments  dex- 
trose, d-mannose,  levulose,  d-galactose,  saccharose  and  maltose. 


240 


FAMILY  OF  SACCHAROMYCETACEAE 


SACCHAROMYCES  VALIDUS.     Hansen 

Saccharomyces  validus,  at  first  called  S.  pastorianus  III  is  a  top 

yeast  isolated  by  Hansen  l 
Q  fj^Ci  $  from  a  bottom  fermentation 


ft  n 

U  ^  m  keer  wnere  ^  caused 
cloudiness.  In  the  vegeta- 
tion at  the  bottom  of  beer 
wort  the  cells  are  ordinarily 
elongated  in  the  form  of 

sausages  intermingled  with 

r 
ment  in  Wort  (after   a  large  or  small  number  of 

Hansen).  round  Qr   oval  cellg      (Fig 

109.)     The  temperature  limits  of  budding  are:    minimum,    0.5°  C., 
maximum  39-40°  C. 

Temperatures  for  the  Formation  of  Ascospores 
At  29°  C.  no  development  of  ascospores. 

27-28°  appearance  of  first  rudiments  in  about  35  hours 


oung  Cells  fromSedi- 


25  50 

OFvQ 

22° 

jyo 

ICO 

10.5° 

O    rtO 


u 


u 


t( 


U 

"       " 

(I  (( 


no          a 

29      " 

44         K 

CQ  « 

7  days 

Q  K 


4°    no  development  of  ascospores. 

The  ascs  are  ellipsoidal  or  elongated.  The  ascospores  measure 
2-4  IJL  in  diameter  and  are  variable  in  number  in  each  asc  (1  to  10) 
(Fig.  110.)  Sometimes  they  germinate  after  having  undergone  a 
copulation  (Marchand)  but  more  often  singly. 

Temperatures  for  Formation  of  Scum 
At  34°  C.  no  formation  of  scum. 


26-28°  at  the  end  of     7-10  days  feeble  development 
20-22°   "    "       "     "     9-12     "         "  " 

13-15°   "    "       "     "  10-12     "         "  " 

6-7°     "    "      "     "     1-2  months    " 
3-5°     "    "      "     a     5-6       "         "  " 

2-3°    no  formation  of  scum. 

1  Hansen,  E.  C.    See  references  under  S.  pastorianus. 


SACCHAROMYCES  ELLIPSOIDEUS 


241 


Microscopic  appearance  of  the  cells  in  the 
scum  follows:  At  20-28°  C.  the  cells  in  the  scum 
have  almost  the  same  shape  as  those  in  the  sedi- 
ment. At  15  to  3°  C.  and  in  old  scums,  one  may 
see  colonies  composed  of  elongated  cells  in  the 
shape  of  sausages  in  which  the  appearance  is 
much  like  a  mycelium.  (Fig  111.)  In  yeast  water 
gelatin,  on  streaks,  one  may  see  at  the  end  of 
sixteen  days  a  very  irregular  border.  This  yeast 
ferments  d-mannose,  levulose,  d-galactose,  sac- 
charose and  maltose. 

LOGOS  YEAST.    Van  Laer  and  Denamur 


Fig.  111.— S.validus. 
Vegetative  Cells 
from  Scum  at  13-3° 
C.  (after  Hansen). 


Here  we  shall  again  mention  a  species  whose 
morphology  is  very  little  known  but  which  is  of 
very  much  importance  industrially.  It  was 
isolated  by  Van  Laer  and  Denamur 1  from  among  the  yeasts  used 
in  the  brewery  of  Logos  and  Company  of  Rio  de  Janeiro  in  Brazil. 
Its  origin  is  unknown;  it  seems  to  have  originated  in  a  spontaneous 
fermentation  of  sugar-cane  juice.  It  has  the  shape  of  S.  pastorianus. 
During  the  fermentation  the  cells  remain  attached  in  large  masses 
which  settle  to  the  bottom  of  the  fermentation  vats.  It  is  a  bottom 
yeast  with  a  slow  fermentation  which  produces  little  alcohol.  It  is 
very  much  attenuated.  It  ferments  dextrine,  inuline,  dextrose,  d-man- 
nose, d-galactose,  levulose,  saccharose,  maltose,  raffinose,  melibiose, 
and  a-methylglucoside. 


Fig.  112.- 
soideus. 


SACCHAROMYCES  ELLIPSOIDEUS.     Hansen 
Syns:  s.  ELLIPSOIDEUS  i.  Hansen.  —  s.  ELLIPSOIDEUS.  Reess 

This  yeast  was  discovered  by 
Hansen2  on  the  surface  of  raisins. 
It  is  a  bottom  yeast  which 
plays  an  important  r61e  in  vini- 
fication.  In  the  sediment  in 
wort  cultures,  it  possesses  ellip- 
tical or  round  cells;  elongated 
cells  are  not  common.  (Fig.  112.) 


•S.ellip- 
Young 


Cells  from  Sedi-  The  temperature  limits  for  bud- 
Wort  (after  Han-  ding  on  beer  wort  are:  minimum, 
sen).  0.5°  C.,  maximum,  40-41°  C. 


Fig.  113.  — Ascs 
ellipsoideus 
Hansen) . 


of  S. 
(after 


1  Van  Laer,   H.,  and  Denamur.     Notice  sur  une  levure  attenuation,    limite 
tres  e"leve"e.     Moniteur  scientific,  1895. 

2  Hansen,  E.  C.    See  references  for  S.  pastorianus. 


242 


FAMILY  OF  SACCHAROMYCETACEAE 


Temperatures  for  Ascospore  Formation 
At  32.5°  no  development  of  ascospores. 

30.5-31.5°  appearance  of  first  rudiments  in  about  36  hours. 


29.5° 
25° 
18° 
15° 

10.5° 

7  50  a  n     a 

4°  no  development  of  ascospores. 


23  " 
21  " 
33  " 
45  " 
41  days 
11  " 


The  ascs  are  ordinarily  ellipsoidal  and  small.  They  enclose  from 
one  to  four  ascospores  which  measure  2  to  5  /z  (Fig.  113).  They 
germinate  after  having  copulated  two  by  two  (Marchaud)  in  about 
half  of  the  cases. 


Temperature  for  Formation  of  Scum 
At  38°  no  formation  of  scum. 

33-34°  at  the  end  of    8-12  days  fully  developed. 
26—28°   (<     ll      ll     ll     Q  I  A     it         u  i( 

20-22°  "     "      "     "  10-17     "         "  " 

13-15°  "     "      "     "  15-30     " 

6-7°     "     "      "     "     2-3  months 

5°  no  formation  of  scum. 


Microscopic    appearance   of   the    cells    in  the 
scum  is  as  follows:   At  20-34°  C.  and  at  6-7°  C., 
the  scum  includes   cells  which  are  small  among 
which  the  sausage-shaped  cells  are  abundant.     At 
13-15°  C.  in  old  scums,  one  may  see  branching 
Fig.    114.  —  S.   ellip-  colonies  with  sausage-shaped  cells,  either  shorter 
cSTfrom^um1!?  elongated.     (Fig.  114.)    In  beer  wort  to  which  has 
13-15°  C.  (after  Han-  been  added  5?   per  cent  of  gelatin,    on    streaks 
at  25°  C.,  one  may  very  closely  separate  the  cells 
of  the  preceding   species.  (S.  cerevisiae,  Pastorianus,  intermedius  and 
validus)  by  a  peculiar  structure  in  the  form  of  a  network  which  is  not 
able  to  escape  the  naked  eye.    This  yeast  ferments  saccharose,  dextrose 
and  maltose. 

TOASTS  RELATED  TO  S.   EWpsoideus 

Numerous  yeasts  are  used  in  the  making  of  wine  which  are  related 
to  S.  ellipsoideus.     They  have  been  described  by  Aderhold,  Hotter, 


SACCHAROMYCES  VINI  MUNTZII  243 

Lendner,    Marx,    Miiller-Thurgan,    Nastjukow,    Osterwalder,   Seifert, 
Wortmann,  Jacquemin,  Kayser  and  Jorgensen. 

The  most  characteristic  are  the  Johannisberg  I  and  II  yeasts,  and 
S.  vini  Muntzii.  We  shall  mention  these  rapidly. 

JOHANNISBERG  YEAST  I.    Wortmann1 

This  species  possesses  round,  oval,  or  pointed  cells.  It  forms  a 
scum  composed  of  oval  cells  at  26-27°  C.  The  ascospores  appear  at 
25-26°  C.  at  the  end  of  28  to  30  hours.  They  germinate  according  to 
Marchaud  after  having  copulated. 

JOHANNISBERG  YEAST  II.    Wortmann 

This  yeast  has  been  described  by  Wortmann  and  Aderhold.2 
It  possesses  oval  cells  longer  than  the  preceding  yeast,  but  never 
pointed.  The  limits  of  temperature  for  budding  on  beer  wort,  accord- 
ing to  Hansen,  are  37-38°  and  0.5°  C.  Ordinarily  it  is  a  bottom 
yeast.  It  sporulates  very  abundantly  on  the  plaster  block.  The 
temperature  limits  for  sporulation  are  2.3°  and  33-34.5°.  The  as- 
cospores are  to  the  number  of  four  in  each  asc.  It  has  been  shown 
that  they  fuse  more  often  two  by  two  before  germinating  and  under- 
going a  true  copulation  (parthenogamy) 3  (Fig.  35).  Germination  is 
accomplished  by  budding  at  some  point  on  the  copulation  canal.  This 
species  forms  a  scum  composed  of  round  or  sausage-shaped  cells. 

SACCHAROMYCES  VINI  MUNTZH.    Kayser 

This  yeast  was  found  by  Kayser 4  on  grapes.  It  is  made  up  of 
cells  in  chains  which  possess  a  vacuolar  protoplasm  and  die  at  about 
55°.  The  ascospores  form  at  the  end  of  about  42  hours  at  25°.  This 

1  Wortmann,  J.    Landw.  Jahrbucher,  XXI,  1892. 

2  Aderhold,  R.    Die  Morph.  der  deutschen  Sacch.  ellipsoideus  Rassen.    Landw. 
Jahrbucher,  XXIII,  1894. 

3  In  this  yeast  the  fusion  of  ascospores  exhibits  very  curious  characteristics. 
In  a  certain  number  of  cases  the  zygospore,  formed  by  the  union  of  two  asco- 
spores, commences  to  germinate  before  nuclear  fusion  has  commenced.     The  two 
nuclei  take  a  position  in  the  middle  of  the  copulation  canal  and  fuse  when  the 
first  bud  forms.     At  the  time  when  nuclear  fusion  takes  place  the  nucleus  which 
results  from  it  quickly  elongates  similar  to  a  bud  and  divides  by  amitosis  in  such 
a  way  as  to  furnish  a  nucleus  to  the  bud.     Sometimes,  however,  nuclear  fusion 
does  not  seem  to  be  accomplished.    The  two  nuclei  join  and  seem  to  divide  simul- 
taneously in  a  manner  to  form  four  nuclei,  two  of  which  remain  in  the  zygospore 
and  the  other  two  enter  the  bud. 

4  Kayser,  E.     Contr.  a  1'etude  des  levures  de  vin.    Ann.  de  PInstitut  Pasteur, 
t.  VI,  1892,  et  les  Levures,  Masson  et  Gauthier-Villars,  editeurs,  2e    ed.,  1905. 


244 


FAMILY  OF  SACCHAROMYCETACEAE 


yeast  ferments  saccharose,  dextrose,  levulose  and  maltose.  It  possesses 
characteristics  of  a  top  yeast.  The  ascospores  according  to  Mar- 
chaud  undergo  a  copulation  just  before  they  germinate. 


SACCHAROMYCES  TURBEDANS.     Hansen 
Syn:  s.  ELLIPSOIDEUS  n.  Hansen. — s.  ELLIPSOIDEUS.  Reess 

Ordinarily  this  is  a  bottom  yeast  which  was 
found  by  Hansen l  in  beer  and  first  described 
under  the  name  of  S.  ellipsoideus  I.  He  found 
a  very  evident  trouble  in  beer  and  this  yeast 
may  be  regarded  as  an  unfavorable  species, 
more  so  than  S.  validus.  In  sediments  in  beer 
wort  it  always  has  round  or  elliptical  cells. 
Elongated  cells  are  rare.  (Fig.  115.)  The 
temperature  limits  for  budding  in  beer  wort 
are:  minimum,  0.5°  C.,  maximum  40°  C. 


Fig.  115.  —  S.  Turbi- 
dans.  Young  Cells 
from  Sediment  in 
Beer  Wort  (after 
Hansen). 


Fig.  115-A.  —  Parasaccharomyces  Ashfordii,  Anderson. 

1,  Cells  from  Young  Beer  Wort  Culture.  —  2,  a,  Moniliform  Clusters  beneath  the  Surface  of  an  Old 
Agar  Plant;  b,  Cells  from  Surface  of  the  Same  Culture.  —  3,  Young  Cells.  —  4,  Old  Cells. 

Temperature  Limits  for  Ascospore  Formation 
At  35°  C.  no  ascospore  formation. 

33-34°  appearances  of  first  rudiments  in  31  hours. 


'  27 
"  23 


'  22 

"  27 

"  42 

"     5J  days 

"    9       " 


33° 

31.5° 

29° 

25° 

18° 

11° 

8° 

4°  no  ascospore  formation. 

1  Hansen,  E.  C.    See  references  under  S.  pastorianus. 


SACCHAROMYCES   WILLIANUS  245 

The  ascospores  are  usually  5  /i  in  diameter  (Fig.  116).  Accord- 
ing to  Marchaud,  they  germinate  after  having  copulated. 

Temperatures  for  Formation  of  Scum 
At  40°  no  formation  of  scum. 

36-38°  at  the  end  of  8-12  hours  feeble  formation. 
33-34°  "     "      "     "  3-4        "          "  " 

2fi— 28°    "     <f       l(      u  4—5         tl  l<  il 

20-22°   "     "      "     "  4-6        "          "  " 

13-15°  "     "      "     "  8-10      "          "  " 

£_7o     „     «      tt     «  1-2  months 

3-5°     "     "      "     "  5-6 
2-3°     no  formation  of  scum. 

The  microscopic  appearance  of  the 
cells  in  the  scum  varies.  At  the  beginning 
the  cells  look  like  those  in  the  sediment 
but,  as  a  rule,  they  are  a  little  longer. 
In  old  scums,  one  may  see  colonies  with 
short  and  long  cells  or  tubular  cells  with 
branching  projections.  We  have  seen  that  Fig.  116.  — Ascs  in  Saccharo- 
Hansen  was  able  to  transform  this  yeast,  mv™*  turbidans  (after  Han- 
normally  a  bottom  yeast,  into  a  top  yeast. 

SACCHAROMYCES  WILLIANUS.    Saccardo l 
Syn:  SACCHAROMYCES  i  OF  WILL.  Bay 2 

The  species  has  been  described  and  named  by  Will,3  with  the 
provisional  name  of  yeast  No.  811.  It  is  a  bottom  yeast  related 
to  S.  turbidans.  Its  cells  are  egg-shaped. 

The  limits  of  temperature  for  the  formation  of  ascospores  on 
plaster  blocks  are  39-41°  C.  and  4-9°  C.  The  optimum  is  34°  C. 
At  this  temperature  sporulation  appears  at  the  end  of  11  hours.  The 
ascospores  measure  1.5  to  5  /*  in  diameter,  more  often  3-5  /z.  They 
appear  at  first  very  refractive,  homogeneous,  and  show  vacuoles  with 
fat  globules.  Their  number  never  exceeds  four  for  each  asc.  They 
germinate  ordinarily  after  having  copulated.  The  temperature  limits  for 
the  formation  on  beer  wort  of  a  scum  are  39-41°  C.  and  4-5°  C.  The 
cells  of  the  scum  are  elongated  and  form  branching  colonies.  The 

1  Saccardo,  P.  A.     Syllage  fungorum.     Padoue,  II,  1895. 

2  Bay,    J.    C.      The  sporeforming  species  of  the  genus   Saccharomyces.    The 
American  Naturalist,  XXVII,  1893. 

3  Will,  H.     Zwei   Hefearten  welche   abnorme  Veranderungen  in  Bier  veran- 
lassen.    Zeitschr.  f.  d.  ges.  Brauw.,  XIV,  1891. 


CH1FOHNIA  COUEtt 

on ARMAGH  - 


246  FAMILY  OF  SACCHAROMYCETACEAE 

colonies  develop  on  wort  gelatin  with  irregular  shapes  and  with  the 
appearance  of  a  network  with  large  meshes.  Later  the  center  be- 
comes compact  with  irregular  contours.  The  thermal  death  point 
for  the  vegetative  cells  is  about  70°  C.  This  species  produces  a  disagree- 
able taste  in  beer  and  causes  very  pronounced  difficulties. 

SACCHAROMYCES  BAYANUS.     Saccardo 
Syn:  SACCHAROMYCES  n  OF  WILL.     Bay 

This  species  was  described  by  Will  at  the  same  time  as  the  pre- 
ceding one.  He  designated  it  provisionally  as  Yeast  II.  It  also  be- 
longs to  the  type  ellipsoideus.  The  cells  have  the  shape  of  pointed 
eggs  about  7  to  11  JJL  long  and  5  to  6  ju  wide.  The  temperature  limits 
for  the  formation  of  ascospores  on  plaster  blocks  are  30-32°  and 
0.5-3°  C.  The  optimum  is  24-25°  C.  At  this  temperature,  the  asco- 
spores appear  at  the  end  of  30  hours.  They  are  to  the  number  of  1 
to  4  per  asc  and  attain  2  to  4  microns  in  diameter.  They  germinate 
usually  after  having  copulated.  In  old  scums,  the  cells  form  budding 
colonies  with  branches.  They  are  able  to  reach  30  microns  in  length 
and  2  to  3  microns  in  diameter.  The  thermal  death  point  for  vegeta- 
tive cells  is  about  70°  C.  This  species  causes  cloudiness  in  beer  and  at 
the  same  time  produces  a  disagreeable  aromatic  odor,  which  is  similar  to 
that  of  decayed  fruit,  and  an  extremely  astringent  taste. 

SACCHAROMYCES  ILICIS.    Gronland 

This  species  was  found  by  Gronland  1  on  the  fruits  Ilex  aquifo- 
lium.  It  is  a  bottom  yeast  with  generally  spherical  cells.  The  tem- 
perature limits  for  sporulation  are  8-9.5°  and  36-38°.  The  ascospores 
are  devoid  of  vacuoles.  The  scum  contains  cells  which  are  slightly 
elongated.  Streak  cultures  on  gelatin  have  a  farinaceous  appearance. 
In  beer  wort  this  yeast  produces  about  2.8  per  cent  of  alcohol  by 
volume.  Its  vegetation  gives  a  very  disagreeable  taste. 

SACCHAROMYCES  AQUIFOLH.    Gronland 

This  yeast  was  also  found  by  Gronland  in  the  fruit  Ilex  aquifo- 
lium.  It  is  a  top  yeast  with  large  round  cells.  The  temperature  limits 
for  the  formation  of  ascospores  are  8°-10.5°  and  from  27.5°  to  31°. 
The  ascospores  possess  vacuoles.  The  scum  is  made  up  of  spherical 
and  oval  cells.  Streak  cultures  on  gelatin  have  variable  appearances. 
This  species  in  wort  gives  a  very  disagreeable  taste.  It  produces  in 
wort  about  3.7  per  cent  of  alcohol  by  volume. 

1  Gronland,  C.  En  ny  Torula-Art  og  to  nye  Saccharomyces-Arter  Vidensk. 
Med.  fra  den  Naturh.  Foren,  Copenhagen,  1892. 


SACCHAROMYCES  SAKfi  247 

SACCHAROMYCES  PIRIFORMIS.    Marshall-Ward 

Ward  l  isolated  this  yeast  from  ginger  beer.  It  grows  in  symbiosis 
with  Bacterium  vermiforme  and  is  found  in  the  sheath  of  this  organism. 
The  bacterium  seems  to  destroy  certain  substances  which  are  detri- 
mental to  the  yeast.  This  yeast  possesses  ellipsoidal  or  round  cells 
like  those  of  Saccharomyces  ellipsoideus.  The  temperature  limits  for 
budding  are  35°  and  10°  C.  It  sporulates  on  plaster  blocks  at  the  end 
of  24  hours  at  25°  C.  Ordinarily  the  ascs  contain  4  ascospores.  It 
causes  an  active  fermentation  in  saccharose  solutions  and  gives  a 
white  waxy  sediment.  In  beer  wort,  it  produces  only  a  feeble  fer- 
mentation and  gives  a  scum  made  up  of  cells  shaped  like  pears  or 
small  sausages. 

SACCHAROMYCES  VORDERMANNII. 

Went  and  Prinsen-Geerligs 

This  yeast  was  discovered  by  Went  and  Geerligs 2  in  a  ferment 
used  in  Java  for  the  manufacture  of  arrack.  The  cells  are  ellipsoidal 
in  the  form  of  an  egg  or  onion.  The  ascs  enclose  four  ascospores. 
This  species  produces  no  scum  in  sugar  solutions,  but  simply  a  ring. 
It  yields  about  10  per  cent  of  alcohol.  Saccharomyces  vordermannii 
is  the  essential  agent  in  the  fermentation  of  arrak. 

SACCHAROMYCES  SAKE.    Yabe3 
Syn:  SAKE  YEAST.    Kosai 4 

This  yeast  is  used  by  the  Japanese  in  the  preparation  of  Sake  from 
rice.  The  saccharification  of  the  starch  is  accomplished  by  Rhizopus 
oryzae.  The  sugar  thus  obtained  is  finally  decomposed  to  alcohol 
and  C02  by  means  of  the  Saccharomyces  sake.  This  yeast  possesses 
spherical  cells  6  to  12  JJL  in  diameter.  Ascospores,  6  to  12  microns  in 
diameter,  are  secured  on  plaster  blocks  at  3-4°  C.  (minimum)  and  in 
36  hours  at  40°-41°  C.  (maximum),  in  40  hours  at  30-32°  C.  It  easily 
ferments  saccharose,  maltose,  levulose,  dextrose,  d-mannose,  and 
a-methylglucosides  and  with  difficulty  trehalose  and  d-galactose.  It 
decomposes  raffinose  into  melibiose  and  levulose  but  does  not  hydro- 
lize  melibiose. 

*.Ward,  M.  The  ginger  beer  plant  and  the  organisms  composing  it.  Philos. 
Trans.  Royal  Society,  183,  1898. 

2  Went,   F.,    and  Prinsen-Geerligs.     Beobachtungen  iiber  die  Hefearten  und 
zuckerbildenen  Pilze  der  Arrakfabrikation  Verhand.  d.  Konigl.    Akad.  d.  Wetensch. 
te  Amsterdam,  4,  1895. 

3  Yabe,  K.     Ueber  den  Ursprung  von  Sakehefe.     Imperial  University  College 
of  Agriculture  Bulletin,  3,  1897. 

4  Kosai,  Y.     Chemie  und  biologic  Unters.,  iiber  Sakebereitung.     Cent.  Bakt. 
1,  1900. 


248  FAMILY  OF  SACCHAROMYCETACEAE 


SACCHAROMYCES  CARTILAGINOSUS.     Lindner1 

This  species  was  found  by  Matthes  in  kephir  grains.  It  gives  a 
smoky  taste  to  wort.  Its  cells  possess  a  very 
granular  protoplasm.  The  ascs  contain  3  to 
4  ascospores  (Fig.  117).  On  beer  wort,  S. 
cartilaginosus  forms  at  the  end  of  a  few  weeks, 
on  the  surface  of  the  liquid,  small  floating 
colonies  of  a  firm  consistency  —  almost  car- 
tilaginous. These  unite  and  increase  in  size 
and  result  in  making  a  scum.  The  yeast 

Fig.    117.  —  Saccharomyces  sediment  is  flocculent.    The  giant  colonies  are 
cartilaginosiJis.  Cells  from    f  ,  ,    ,      ,_.,  .  ,  -  ,  , 

Young  Culture  on  Beer  folded.    This  yeast  ferments  dextrose,  d-man- 

Wort,    and   Ascs   (after   nosej    levulose,    saccharose   and   maltose   but 
produces  only  feeble  fermentation  in  d-galac- 
tose.     It  may  also  have  an  action  on  raffinose. 


SACCHAROMYCES  BATATAE.    Saito 

This  yeast  was  isolated  by  Saito 2  from  moromi,  a  fermentable  dough, 
made  of  a  mixture  of  "  koji  "  and  potato  (boiled)  which  is  used 
in  the  manufacture  of  Brandevin  (a  wine  in  Japan).  This  yeast  is 
the  most  active  agent  in  this  fermentation.  The  cells  are  oval  and 
elliptical.  In  the  scums  they  often  have  the  shape  of  cells  of  Sac- 
charomyces pastorianus.  The  ascospores  form  at  the  end  of  24  hours 
at  25°  C.  They  are  spherical,  very  refractive  and  to  the  number  of  2 
or  3  in  each  asc.  In  beer  wort  at  25°  C.  this  species  produces  3  per 
cent  of  alcohol  by  volume.  Saccharomyces  batatae  easily  ferments  dex- 
trose, levulose,  saccharose  and  maltose,  more  difficultly  d-galactose, 
and  raffinose;  it  has  no  action  on  melibiose,  lactose,  inuline  and 
a-methylglucosides. 

SACCHAROMYCES  MULTISPORUS.     Jorgensen.3 

This  is  a  wild  yeast  isolated  by  Holm  from  an  English  top  yeast. 
Most  of  the  cells  are  ellipsoidal.  However,  a  large  number  are  large 
round  cells.  These  latter  as  well  as  the  ellipsoidal,  are  capable  of 
forming  ascs.  Spores  appear  on  plaster  blocks  at  the  end  of  40  hours 

1  Lindner,  P.     Mikroskopische  Betriebskontrolle  in  den  Garungswerben,  Paul 
Parey,  Berlin,  6th  edition,  1909. 

2  Saito,   K.     Mikro.  Studien  ttber  die  Zubereitung  der  Betatenbranntweins. 
Cent.  Bakt.  18,  1907. 

3  Jorgensen,  A.     Die  Mikroorganismen  der  Garungsindustrie.      5th  edition, 
Paul  Parey,  Berlin,  1909. 


CIDER  YEASTS   OF  PEARSE   AND   BARKER         249 

at  25°  C.  The  ascs  formed  by  the  ellipsoidal  cells  form  only  three  or 
four  ascospores;  those  from  the  large  cells  form  nine  or  ten.  The 
ascospores  are  round  and  very  refractive.  After  long  culturing  in 
must  sugar  solutions,  this  yeast  loses  its  ability  to  sporulate.  Sac- 
charomyces  multisporus  is  a  bottom  yeast  which  adheres  closely  to 
the  culture  flask,  so  well  that  it  is  difficult  to  detach  it.  A  thin  scum 
is  formed.  It  yields  about  4  per  cent  of  alcohol  by  volume  and  pro- 
duces a  disagreeable  taste.  This  yeast  ferments  dextrose,  maltose, 
and  saccharose. 

SACCHAROMYCES  MALI  RISLERL     Kayser1 

Discovered  in  specimens  of  cider  by  Kayser,  this  yeast  possesses 
spherical  cells,  from  4  to  6  microns  in  diameter,  which  have  a  thermal 
death  point  of  60°  C.  Oil  liquid  media,  they  produce  an  adhesive 
deposit  on  the  walls.  The  ascospores  form  at  15°  C.  at  the  end  of 
ninety  hours.  This  species  ferments  saccharose,  dextrose  and  maltose. 


CIDER  YEASTS   OF  PEARSE  AND  BARKER 

These  species  have  been  isolated  by  Pearse  and  Barker2  from 
ciders  in  Alford  and  Kingston,  England. 

Yeast  A.  The  cells  in  beer  wort  are  usually  oval  while  in  old 
cultures  they  become  elongated.  The  cells  developing  on  gelatin 
sometimes  take  the  form  of  sausages.  The  maximum  temperature 
for  budding  is  situated  between  35°  and  38°  C.  Spores  are  easily  formed 
in  90  hours  on  potato  and  porous  porcelain  at  room  temperature. 
They  commence  toward  15°  and  stop  at  26°  C.  They  are  often  ob- 
served also  in  old  cultures  on  wort.  The  ascospores  measure  3.1 
microns  in  diameter.  At  the  time  of  germination  the  wall  of  the  asc 
is  ruptured,  the  ascospores  swell  and  germinate  by  normal  budding. 
On  gelatin  this  yeast  forms  dry  spherical  colonies,  with  slightly  in- 
dented border.  On  streaks,  it  produces  a  creamy  vegetation,  with 
folded,  slightly  fringed  borders.  It  liquefies  gelatin  very  slowly.  This 
species  ferments  saccharose,  dextrose,  levulose  and  maltose. 

Yeast  B.  This  yeast  is  much  like  the  preceding  one  in  which  the 
cells  have  the  same  shape.  Their  dimensions  vary  between  6.8  and 
10.2  to  4.4  ju.  The  maximum  temperature  for  budding  is  around  33°  C. 
Sporulation  is  accomplished  easily  on  carrot  and  on  porous  porcelain. 
It  appears  on  this  last  substrate  in  about  42  hours  at  26°  and  in  90 

1  Kayser,  E.    Etude  sur  la  fermentation  du  cidre.    Ann.  Inst.  Pasteur,  4,  1890. 

2  Pearse,  B.,  and  Barker,  P.    The  yeast  flora  of  bottled  ciders.    Jour.  Agricul- 
tural Science,  3,  1908. 


250  FAMILY  OF  SACCHAROMYCETACEAE 

hours  at  room  temperature.  It  begins  at  14°  C.  It  is  also  observed  in 
old  cultures  on  gelatin.  The  ascospores  are  about  3.9  ju  in  diameter. 
Germination  is  accomplished  as  in  Yeast  A.  This  species  ferments 
dextrose,  levulose,  saccharose  and  maltose. 

Yeast  H.  This  species  on  beer  wort  has  oval  cells,  which  in  old 
cultures  may  elongate.  The  maximum  temperature  for  budding  is 
situated  between  30°  and  32°  C.  Sporulation  is  accomplished  easily 
on  porous  porcelain,  potato,  carrot  and  on  wort  gelatin.  The  asco- 
spores are  to  the  number  of  two,  three  or  four  per  asc.  They  have  a 
diameter  of  2.3  JJL.  Their  germination  seems  to  be  accomplished  by 
parthenogamy.  On  gelatin,  the  colonies  are  white,  dry,  spherical  and 
on  streaks  the  vegetation  is  moist  with  fringed  borders.  Gelatin  is 
liquefied  at  the  end  of  some  time.  This  yeast  ferments  dextrose,  levu- 
lose, maltose  and  saccharose. 

Yeast  I.  The  cells  are  oval  in  the  form  of  a  sausage  with  granules. 
The  maximum  temperature  for  budding  is  between  35°  C.  and  38°  C. 
Sporulation  is  accomplished  rapidly  on  gelatin.  On  plaster  blocks  at 
the  end  of  22  hours  it  appears  at  26°  C.  The  ascospores  measure  3.5/z 
in  diameter  and  vary  from  2  to  4  per  asc.  Germination  begins  by 
swelling  of  the  ascospore  which  ruptures  the  cell  wall  and  normal 
budding  takes  place.  The  colonies  on  gelatin  plates  have  the  appear- 
ance of  cones  with  furrows  on  the  surface  and  fringed  edges.  On 
streaks,  the  vegetation  is  creamy  and  moist  with  irregular  borders. 
This  species  ferments  dextrose,  levulose,  maltose  and  saccharose. 

Yeast  K.  This  species  was  found  in  the  black  Kingston  cider.  The 
cells  are  oval  or  sausage  shaped.  The  maximum  temperature  for 
budding  is  around  38°  C.  Sporulation  is  accomplished  on  porous  por- 
celain at  26°  C.  The  ascospores  are  to  the  number  of  2  to  4  per  asc. 
Their  diameter  is  around  3.9  ju.  The  colonies  on  gelatin  plates  are 
spherical  and  dry  with  thin  edges.  This  species  ferments  dextrose, 
levulose,  maltose,  and  saccharose. 

SACCHAROMYCES  TOKYO.    Nakazawa 

This  yeast  was  isolated  by  Nakazawa  l  from  the  fermentation  of 
Sake".  It  has  spherical  cells  (1.2  to  3.2  JJL)  or  elliptical  cells  (3.0-14.0  jj, 
by  2.0  to  9.0  /x)  often  intermixed  with  large  cells,  ovoid  or  pear  shaped. 
The  protoplasm  contains  few  or  no  granules.  The  ascospores,  of  which 
the  number  varies  from  one  to  four  per  asc,  form  in  24  hours  at  35°  C. 
The  optimum  temperature  for  the  formation  of  ascospores  is  in  the 
vicinity  of  31°  C.  The  ascospore  appears  in  about  16  hours.  The 
minimum  is  about  10°  C.  The  scum  is  formed  with  difficulty.  When 

1  Nakazawa,  R.    Zwei  Saccharomyceten  aus  SakShefe.    Cent.  Bakt.  22,  1909. 


SACCHAROMYCES  FROM  SHIRO-KOJI  251 

cultivated  in  yeast  water  containing  5  per  cent  of  saccharose  this 
yeast  gives  a  reddish  color.  Giant  colonies  are  formed  on  gelatin  wort. 
Saccharomyces  Tokyo  seems  to  be  a  bottom  yeast  causing  rapid  fer- 
mentation. It  ferments  dextrose,  saccharose,  d-galactose  and  mal- 
tose; however  it  has  no  action  on  melibiose  nor  lactose. 


SACCHAROMYCES  YEDDO.     Nakazawa 

This  yeast,  related  to  the  preceding  one,  was  also  isolated  by 
Nakazawa1  from  Sake  fermentation.  It  possesses  spherical  (3.2  to 
6.4  ju)  or  ellipsoidal  cells;  often  the  cells  are  sausage  shaped.  Giant 
cells  are  often  found.  The  protoplasm  is  homogeneous  and  contains 
few  or  no  granulations.  In  neutral  yeast  water,  with  5  per  cent  sac- 
charose, this  yeast  imparts  a  yellowish  red  coloration  to  the  fluid. 
It  produces  ascospores  to  the  number  of  1  to  4  per  asc.  •  The  maxi- 
mum temperature  for  the  formation  of  ascospores  is  about  35°  C. 
The  ascospores  are  formed  in  about  18  hours.  The  optimum  tempera- 
ture is  around  31°  C.  At  this  temperature  the  ascospores  appear  in 
about  14  hours.  The  minimum  temperature  is  between  14°  and  10°  C. 
Saccharomyces  Yeddo  forms  a  thick  shiny  scum,  in  which  the  colora- 
tion varies  from  a  white  to  a  yellow.  It  is  a  bottom  yeast  and  a  slow 
fermenter.  It  ferments  dextrose,  saccharose,  d-galactose  and  maltose 
but  has  no  action  on  melibiose,  nor  lactose. 


SACCHAROMYCES  FROM   SHIRO-KOJI.    Saito 

This  species  was  isolated  by  Saito  2  from  Shiro-Koji.  It  possesses 
globular  isolated  cells,  from  5  to  6  ju,  in  diameter.  The  contents  show 
a  hyaline  protoplasm  with  one  or  many  vacuoles.  The  ascospores  are 
almost  always  to  the  number  of  two  in  each  asc.  They  are  round  and 
measure  2  to  5ju  in  diameter.  The  giant  colonies  appear  as  little  points 
which  form  a  mass  of  yellow  growth  without  folds.  The  cultures 
on  gelatin  as  streaks,  produce  a  liquefaction  of  this  medium.  This 
species  never  produces  a  scum  on  sugar  solutions  but  a  ring  is  secured 
after  fermentation.  It  ferments  dextrose,  levulose,  d-galactose,  sac- 
charose, maltose  and  raffinose  but  has  no  action  on  melibiose,  inu- 
line,  lactose  and  a-methylglucosides.  On  beer  wort,  it  produces  5.24 
per  cent  of  alcohol  after  20  days. 

1  Nakazawa,  R.     Zwei  Saccharomyceten  aus  Sakehefe.     Cent.  Bakt.  22,  Avt. 
II,  1909. 

2  Saito,    K.     Notes  on  Formosan   Fermentation  Organisms.     The   Botanical 
Magazine,  15,  1902. 


252  FAMILY  OF  SACCHAROMYCETACEAE 

SACCHAROMYCES  T  AND  V  OF  LUDWIG  ROSE1 

These  species  were  isolated  from  the  mucous  secretions  of  two 
oaks  in  which  they  were  found  associated  with  Saccharomyces  Lud- 
wigii,  Saccharomyces  apiculatus,  Yeasts  F  and  G  of  Rose,  and  End. 
Magnusii.  They  have  the  same  characteristics  and  are  apparently 
identical.  Their  cells  are  elliptical,  later  becoming  round.  Their 
diameter  is  about  5.5  ju.  These  two  yeasts  form  ascospores  easily  to 
the  number  of  two  or  four  in  each  asc.  The  optimum  temperature 
for  the  formation  of  ascospores  on  plaster  blocks  is  25°  C.  These  species 
seem  to  resemble  yeast  No.  689,  isolated  by  Lindner  2  from  secretions 
of  trees  in  the  Berlin  botanical  garden.  In  wort  they  produce  an  ac- 
tive fermentation  of  the  bottom  type.  They  ferment  dextrose,  d- 
mannose,  d-galactose,  levulose,  saccharose,  maltose,  raffinose  and 
a-methylglucoside. 

B.   Second  Sub-Group 

Yeasts  fermenting  dextrose  and  saccharose  but  having  no  action 
on  maltose  or  lactose. 

SACCHAROMYCES  MARXIANUS.     Hansen 

This  species  was  found  by  Marx  on  grapes  and  described  by  Han- 
sen.3   In  must  it  produces  small  oval  cells  which  resemble  very  much 
S.  exiguus  and  S.  ellipsoideus  (Fig.  118).    How- 
Q£5Q    5?   ft  ever,  they  are  easily  distinguished   from  these 

^cO  Q>^JO  f-^n  C&  ^wo  yeas^s  by  the  fact  that  they  form  colonies 
c^,  of  long  cells,  very  rapidly,  in  the  shape  of  a 
sausage,  and  later  on  flocks  which  float  on  the 
liquid.  These  are  composed  of  cells  having 
from  Sediment  after  the  appearance  of  mycelium  and  resemble  the 
(a^ter  Hansen)661  formations  which  one  observes  in  scums  of 

certain  other  yeasts  (S.  cerevisiae,  Pastorianus 

and  ellipsoideus).  These  colonies  are  formed  of  cells  which  are  easily 
detached  from  their  point  of  connection.  On  gelatin  the  cells  develop 
with  a  true  mycelial  formation  with  cross  walls  resembling  the  my-, 
celium  of  Monilia  Candida  (Fig.  119). 

1  Rose,   L.   Beitrage   zur   Kenntniss   der   Organismen   in   Eichenschleimfluss. 
Inaugural  Dissertation,  University  of  Berlin,  June  25,  1910. 

2  Lindner,  P.     Mikroskopische  Betriebskontrolle  in  der  Garungswerben.     Paul 
Parey,  Berlin,  6th  edition,  1909. 

3  Hansen,  E.  Ch.     Recherches  sur  la  physiologic  et  la  morphologic  des  fer- 
ments alcooliques.     VII.     Action  des  ferments  alcooliques  sur  les  diverses  especes 
de  sucre.    Levures  alcooliques  a  cellules  ressemblant  &  des  Saccharomyces.    C.  R. 
du  lab.  de  Carlsb.  v.  II,  Book  5,  1888;   C.  R.  du  lab.  de  Carlsb.  v.  V,  Book  2, 
1902. 


SACCHAROMYCES  MANDSHURICUS 


253 


The  temperature  limits  for  budding  on  beer  wort  are:  minimum 
0.5°  C.  and  maximum  46-47°  C. 
The  maximum  temperature  of 
sporulation  is  situated  between  32 
and  34°,  the  minimum  temperature 
being  between  4  and  8°.  The  op- 
timum is  between  22  and  25°  C. 
(Klocker  J).  Sporulation  is  effected 
very  easily  and  most  abundantly  in 
yeast  water  with  10%  of  must,  and 
on  plaster  blocks.  The  ascospores 
are  spherical  or  oval  and  measure 
3.5  fji  in  diameter. 

This  species  produces  only  traces 
of  a  scum  which  appears  only  after 
two  or  three  months  of  culturing 
on  must.  Its  scum  is  made  up  of 
ellipsoidal  cells  with  a  few  elongated 
and  sausage  shaped. 

In  beer  wort  this  produces  after 
a  long  time  from  1  to  3  %  of  alcohol 
by  volume. 

It  inverts  and  ferments  saccharose.  It  also  ferments  dextrose, 
d-mannose,  d-galactose,  levulose,  raffinose  and  inuline,  but  it  does 
not  act  on  melibiose. 


Fig.  119.  —  Saccharomyces  Marxianus. 
Formation  of  Mycelium  in  Gelatin  to 
which  Yeast  Water  Has  Been  Added 
(after  Hansen) . 


SACCHAROMYCES  MANDSHURICUS.    Saito2 
Saito  isolated  this  yeast  from  Chinese  yeast  used  in  the  making 
of  Sorgho,  an  alcoholic  drink  of  Manchuria.     He  isolated  Saccharomyces 


Fig.  11&-A. — Saccharomyces  mandshuricus. 
Cells  from  Sediment  in  Wort  at  28°  C.;  An 
Asporogenic  Species  (Saito) . 


Fig.  119-B.  —  Saccharo- 
myces mandshuricus. 
Cells  from  Sediment  in 
Beer  Wort  at  28°  C. 
(Saito). 


1  Klocker,  A.     Rech.  sur  les  S.  Marxianus,  apiculatus  et  anomalus.     C.  R. 
des  trav.  du  lab.  de  Carlsberg,  v.  IV,  1895. 

2  See  reference  for  Zygosaccharomyces  Mandshuricus. 


254  FAMILY  OF  SACCHAROMYCETACEAE 

mandshuricus  I,  II,  III,  and  IV.  The  cells  are  oval  or  globular  (6-8) 
in  diameter.  On  gelatin  large  white  round  colonies  are  obtained.  In 
their  centers,  there  is  a  sort  of  crater  with  canals  running  out  around 
the  periphery.  On  beer  wort,  a  scum  is  formed  after  a  time.  The 
spores  are  globular  (2.7-4)  and  on  Gorodkowa's  gelatin  medium 
they  germinate  by  ordinary  budding.  The  temperature  limits  for 
sporulation  are  11°  C.  and  38°  C.  This  yeast  ferments  levulose,  dex- 
trose, mannose,  galactose,  maltose,  saccharose  and  raffinose.  Saccha- 
romyces  mandshuricus  II,  III  and  IV  are  very  closely  related  to  this 
species. 

SACCHAROMYCES  EXIGUUS.     Reess-Hansen 

This  species  was  found  by  Hansen  l  in  pressed  yeast.  It  develops 
in  must  with  a  vegetation  resembling  8.  exiguus  described  by  Reess. 
It  is  not  possible  to  separate  it  with  a  certainty  from  this  latter  yeast. 

The  cells  are  small  and  resemble  the  cells  of  S.  Marxianus,  but 
they  never  give  the  mycelial  formation.  They  do  not  contain  gly- 
cogen. 

The  formation  of  ascospores  and  scums  is  not  very  abundant.  On 
the  contrary,  this  yeast  forms  rings  on  the  culture  tube.  The  cells 
of  the  scum  resemble  those  of  the  sediment.  However,  small  cells 
and  short,  tubular  forms  are  much  more  frequent. 

When  cultivated  in  must  this  S.  exiguus  produces  only  feeble 
quantities  of  alcohol.  It  does  not  cause  any  disease  in  beer.  It  fer- 
ments saccharose,  dextrose,  levulose,  raffinose,  dextrin  and  sometimes 
inuline,  but  it  does  not  act  on  maltose  and  d-mannose  or  melibiose. 


SACCHAROMYCES  ZOPFII.     Artari 

This  yeast  was  found  during  the  manufacture  of  sugar  in  Saxony. 
Since  that  time  it  has  been  found  by  others  in 
samples  of  syrup  in  the  making  of  sugar.  Owen2 
stated  that  this  yeast  was  the  principal  agent  causing 

a    deterioration   of    the    product.    Browne    isolated 
r  ig.       120.  —  oac-  ~ 

charomyces  Zopfii   several  varieties  of  yeasts  from  Cuban  raw  sugar. 

(after  Lindner).      These  are  described  elsewhere. 

1  Hansen,  E.  C.     Recherches  sur    la  physiologic  et    la  morphologic  des  fer- 
ments alcooliques.     VII.     Action  des  ferments  alcooliques  sur  les  diverses  especes 
de  sucre.    Levures  alcooliques  a  cellules  ressemblant  &  des  Saccharomyces.     C.  R. 
du  lab.  de  Carlsb.,  v.  II,  Book  5,  1888;   C.  R.  du  lab.  de  Carlsb.,  v.  V,  Book  2, 
1902. 

2  Owen.     The  occurrence  of  Saccharomyces  Zopfii  in  cane  syrups  and  varia- 
tion in  the  resistance  to  high  temperatures  when  grown  in  solutions  of  varying 
density.    Cent.  Bakt.  Abt.  II,  39,  1913. 


SACCHAROMYCES  COREANUS  255 

This  yeast  has  been  isolated  from  the  manufacture  of  sugar  in 
Saxony.1  It  is  composed  of  short  ellipsoidal  or  spherical  cells  in 
which  the  diameter  may  reach  from  3  to  6  /LI,  exceptionally  8  /z  (Fig. 
120).  When  the  species  is  cultivated  in  a  solution  of  dextrose  with 
5  to  8%  ammonium  sulphate  added,  it  produces  walled  cells.  The 
maximum  temperature  for  budding  in  must  is  33-34°  C.,  the  optimum 
is  28-29°  C.  Sporulation  is  easily  accomplished  as  well  on  liquid  media 
as  on  solid  media.  The  maximum  temperature  for  the  formation  of 
ascs  is  about  32  and  29°  C.  Ascospores  commence  to  appear  at  this 
temperature  at  the  end  of  21  hours.  The  ascospores  are  spherical 
and  measure  from  1.5  to  3/z  in  diameter.  Their  number  is  ordinarily 
two  per  asc,  but  it  may  vary  from  one  to  four.  The  vegetative  cells 
are  able  to  resist  a  temperature  of  130°  dry  heat  for  a  half  hour,  and 
from  66-67°  moist  heat.  According  to  Owen  this  yeast  is  able  to 
resist  90°  C.  for  10  minutes  which  would  locate  the  thermal  death  point 
at  90°. 

SACCHAROMYCES  COREANUS.    Saito 

This  species  was  isolated  by  Saito  2  from  Koji  from  Korea.     The 
cells  are  spherical,  oval  or  sausage  shaped,  and  possess  a  very  re- 
sistant wall.     Their  average  dimension  is  from  3-7  /-(.     The  contents 
are  homogeneous   and   hyaline,  sometimes 
with  large  vacuoles.     The  cells  dissociate 
rapidly  in  such  a  way  that  they  often  appear 
isolated. 

This  yeast  forms  ascospores  very  easily 
on  plaster  blocks.  The  temperature  limits 
for  sporulation  are:  minimum  18-20°;  op- 
timum  31-34°;  and  maximum  toward  35- 


36°  C.  Vegetation   from  Sediment; 

T,     -,  f  £  b,  Ascs  (after  Saito). 

Each  asc   possesses   from   one  to   four 

ascospores,  most  of  them  having  two  to  four  (2  to  3.5  /*  in  diameter). 
The  ascospores  germinate  by  ordinary  budding. 

S.  coreanus  produces  a  moist  scum  in  sugar  solutions  at  25°.  It 
ferments  dextrose,  levulose,  saccharose,  d-galactose,  melibiose  and 
ramnose,  but  has  no  action  on  maltose,  lactose,  arabinose,  inuline 
or  dextrin. 

Giant  colonies  develop  on   decoction  of  Koji  gelatin  and  have  a 

1  Artari,  A.     Ueber  einen  im  Safte  d.  Zuckerfabriken  in  Gemeinschaft  mit 
Leuconostoc  schadlich  auftretenden,  den  Zucker  zu  Alkohol  u.  Saure  vergarenden 
Saccharomyces  (S.  zopfii).    Abh.  d.  naturf.  ges.  zu  Halle,  v.  XXI,  1897. 

2  Saito,   K.     Notizen  iiber  einige  Koreanische  Garungsorganismen.     Centr.  f. 
Bak.,  v.  XXVI,  1910. 


256  FAMILY  OF  SACCHAROMYCETACEAE 

grayish  white  color.  The  center  is  slightly  concave  with  a  surround- 
ing surface  of  radial  bands:  The  edge  is  very  much  indented.  On  Koji 
gelatin  in  plates,  the  colonies  are  punctiform  moist  with  a  grayish- 
white  color.  The  gelatin  is  not  liquefied.  In  streaks  on  the  same 
medium  this  yeast  furnishes  a  thick  white  growth  with  an  indented 
border. 

SACCHAROMYCES  COREANUS.    Forma  Major.    Saito 

Isolated  under  the  same  conditions  as  the  preceding  one,  this 
yeast  is  scarcely  distinguishable  by  its  dimensions,  which  are,  however, 
a  little  larger.  The  cells  are  spherical  or  oval  (8  to  12  /z  in  diameter) 
(Fig.  121,  a)  and  cause  the  formation  sometimes  of  short  mycelial  forma- 
tions. The  ascospores  are  2  to  4  JJL  in  diameter  (Fig.  121,  b).  This 
yeast  forms  no  scum  on  a  decoction  of  Koji. 

SACCHAROMYCES   JORGENSENII.    Laser** 

This  yeast  was  described  by  Lasche.1  It  possesses  small  round 
or  oval  cells  (2.5  to  5/u).  The  optimum  temperature  for  sporulation 
is  25°  C.;  the  temperature  limits  are  8°-12°  and  26°-30°.  At  this 
higher  temperature,  vegetation  rapidly  disappears.  The  ascospores 
are  spherical  and  very  refractive.  They  are  present  ordinarily  to 
the  number  of  2  or  3,  rarely  4,  per  asc.  No  scum  formation  has 
been  noticed.  In  old  cultures,  one  may  observe  a  scant  ring  formation 
resembling  that  of  a  brewery  yeast.  Gelatin  is  slowly  liquefied.  Cul- 
tures on  streaks  have  a  gray  color  with  a  regular  edge. 

C.     Third  Sub-Group 

Yeasts  fermenting  dextrose  and  maltose  but  having  no  action 
on  saccharose  or  lactose. 

SACCHAROMYCES  ROUXII.    Boutroux2 

This  yeast  was  discovered  in  the  juice  of  certain  fruits.  Its  cells 
are  small,  4  to  5  p  in  diameter.  They  are  spherical  or  ellipsoidal 
and  often  arranged  in  chains.  The  ascs  contain  from  1  to  3  ascospores. 
They  are  produced  especially  on  nutrient  fluids.  This  species  pro- 
duces no  extended  scum  but  simply  small  floating  islands. 

1  Lasche*,  A.     Saccharomyces  Jorgensenii.     Der  Braumeister,   Chicago,   1892, 
and  Zeitschr.  f.  d.  ges.  Brauw.  15,  1892. 

2  Boutroux,    L.      Sur   1'habitat   et  la    conversation    des   levures    spontanees. 
Bull,  de  la  soc.  Linn,  de  Normandie,  Vol.  VII,  1883;   Ann.  de  1.  sc.  nat.  Botan. 
17,  1884. 


YEAST  FROM   PULQUE  NO   2. 


257 


YEAST  FROM  PULQUE  NO.   2.     Guilliermond 1 

This  yeast  was  isolated  from  the  fermentation  of  Pulque,  an  al- 
coholic drink  prepared  in  Mexico. 
In  beer  wort  it  develops  as  a  very 
abundant  sediment  with  a  whitish 
yellow  color.  A  wine  fermentation  is 
produced  in  beer  wort  and  a  very 
evident  cloudiness.  After  a  certain 
time,  it  forms  small  floes  with  a 
sparkling  appearance  which  float  in 
the  medium.  Ring  or  scum  forma- 
tions have  not  been  observed.  The 
sediment  is  made  up  of  cells  with 
variable  shapes,  either  oval,  round, 
or  elongated,  with  pointed  ends 


which  resemble  somewhat  S.  Lud-  Fig.  121-A.  -  Yeast  from  Pulque  No. 
wigii.  In  general,  budding  is  ac-  2.  Sediment  in  Beer  Wort  after  15 
complished  at  both  ends  of  the  cells. 

Often  many  buds  are  formed  at  the  same  time  at  each  end,  as  in  the 
yeast-like  structures  of  the  Dematium.  Even  round  cells  budding  like 
Torula  may  also  be  observed.  After  a  few  days  there  is  a  produc- 
tion of  a  deposit  in  the  flask  and  a  true  mycelium  which  buds  like 


Fig.  121-B.  —  Copulated  Ascospores 
Changing  into  Ascs  after  having 
Developed  Cells  in  Yeast  from 
Pulque  No.  2. 


Fig.  121-C.  —  Copulation  of  Asco- 
spores and  Their  Germination  in 
Yeast  from  Pulque  No.  2  (after 
Guilliermond). 


a  Monilia.  The  maximum  temperature  for  growth  is  situated  near 
40°  C.  and  the  optimum  near  29°-30°  C.  The  spores  are  formed  very 
easily  and  abundantly  on  most  solid  media.  They  appear  not  only 
in  the  yeast-like  cells  but  also  in  cells  in  the  mycelium.  Generally, 
they  are  to  the  number  of  four  per  cell.  The  maximum  temperature 
for  sporulation  is  near  37°  C.,  the  optimum  near  25°  C. 

1  Guilliermond,    A.      Levaduras   del    Pulque.    Boletin   de   la    Direcci6n   de 
Estudios  Biol6gicos,  Mexico,  1917. 


258 


FAMILY  OF  SACCHAROMYCETACEAE 


Germination  of  the  spores  is  always  similar  to  that  in  S.  Lud- 
wigii.  The  spores  fuse  two  by  two  by  a  copulation  canal  and  later 
germinate  by  ordinary  budding.  It  often  happens,  in  unfavorable 
solid  media,  that  the  spores  after  having  united  two  by  two,  change 
immediately  into  normal  ascs 


Fig.  121-D.  —  Germination 
and  Copulation  of  Spores 
in  Yeast  from  Pulque  No.  2. 


Fig.  121-E.  —  Ascs  from  Yeast  from  Pulque 
No.  2  on  Beer  Wort  Agar  at  30°  C. 


This  yeast  inverts  and  ferments  saccharose.  It  is  curious  and 
aberrant  having  certain  analogies,  in  the  formation  of  its  mycelium 
and  other  characteristics,  to  Saccharomyces  Ludwigii. 


ZYGOSACCHAROMYCES  SOJA.    Takahashi  and  M.  Yukawa1 
Syn.:    SACCHAROMYCES  SOJA.    Saito 2 

This  yeast  was  isolated  during  the  early  stages  of  ripening  of 
"Shoju  Moromi,"  and  seems  to  be  an  important  species  for  "Shoju  " 
manufacture.  Excepting  the  fermentability  of  galactose,  Saccharo- 
myces soja  seems  to  be  similar  to  this  yeast;  moreover,  there  is  not  a 
great  difference  between  Torula  "Shoju  "  and  this  yeast.  According 
to  Saito's  illustration  it  is  questionable  that  he,  who  gave  the  name 
of  Zygosaccharomyces  japonicus  to  this  "Shoju  "  film  yeast,  comprised 
his  "Shoju "  yeast  into  the  genus  of  Saccharomyces.  Jorgensen 3 
also  has  the  same  inference  about  this  question. 

In  "Koji  "  extract  or  wort  after  5  days  at  20°  C.  the  young  cells 
are  commonly  spherical  or  oval,  3.5-8  jut  in  diameter.  The  contents 
are  homogeneous  and  sometimes  exhibit  vacuoles,  and  are  rich  in 
glycogen.  The  cells  of  old  cultures  in  "Koji"  extract  or  wort  after 

1  Takahashi,   and  Yukawa  M.     Original    communications,    Eighth   Internatl. 
Congress  of  Applied  Chemistry,  v.  XIV,  1912,  p.  166. 

2  Saito,  K.     Cent.  f.  Bak.  II,  Ab.  XVII,  1906. 

3  Jorgensen.     Die  Microorganismen  d.  Garungsindustrie.     IV.   Aufl.   Jorgen- 
sen, 370. 


ZYGOSACCHAROMYCES  SOJA  259 

2-6  months  have  already  been  described,  and  are  almost  the  same  as 
in  Zygosaccharomyces  major. 

On  "Koji  "-extract-gelatin-plate  this  yeast  forms  bright  pearly, 
grayish  white,  mostly  round,  and  elevated  colonies.  On  "Koji  "- 
extract  agar  streak  at  27°  C.  it  forms  a  grayish  white,  waxy,  elevated 
surface,  but  after  a  month  it  becomes  somewhat  brownish  and  the 
center  of  the  growth  becomes  flat.  The  edge  shows  tooth-like  en- 
gravings. In  glucose  "  Sake  "  agar  the  growth  is  yellowish  white, 
of  waxy  luster,  and  forms  an  elevated  smooth  surface  with  fine  stream- 
ing lines.  The  edge  is  somewhat  uneven.  On  stab  the  growth  is  the 
same  as  with  the  preceding  species,  but  the  surface  of  the  isle  is  more 
concent rical.  In  fluid  culture  the  appearance  of  development  of  this 
species  is  very  similar  to  that  of  Zygosaccharomyces  major.  This 
species  can  also  reproduce  and  ferment  in  every  nutrient  fluid  which 
contains  20%  NaCl. 

This  yeast  ferments  dextrose,  levulose,  maltose,  mannose,  but  does 
not  ferment  saccharose,  raffinose,  galactose,  lactose,  a-methylglucoside. 

Formation  and  germination  of  spores  in  this  species  have  already 
been  described  fully.  The  form  and  size  of  spores  are  similar  to  those 
of  Zygosaccharomyces  major,  but  the  numbers  of  sporogenic  cells  are  al- 
ways less  than  in  the  latter  species.  Moreover,  the  time  required  for 
the  occurrence  of  sporulation  is  longer  than  that  of  Zygosaccharomyces 
major. 

This  species  does  not  ferment  saccharose  but  Zygosaccharomyces 
major  attacks  the  same  sugar  quickly  and  both  species  are  easily  dis- 
tinguished from  each  other  by  dimensions  of  the  cells  and  the  growths 
on  glucose- "  Sake  "-agar. 

This  species  differs  from  Zygosaccharomyces  Barkeri  by  the  sporo- 
genic point  of  view  and  the  behavior  toward  maltose,  and  from 
Zygosaccharomyces  priorianus  by  the  cell  forms  of  young  cultures 
and  the  circumstance  of  sporulation.  Zygosaccharomyces  javanicus 
is  easily  distinguished  from  our  yeast  by  the  size  of  cell  and  the  fer- 
mentability  of  galactose,  and  the  formation  of  large  numbers  of  spores 
on  agar.  Zygosaccharomyces  lactis  a  ferments  lactose  but  not  maltose. 
Zygosaccharomyces  japanicus  produces  easily  a  particular  film  on  the 
surface  of  nutrient  fluid.  Both  Zygosaccharomyces  fusoriens  and 
Zygosaccharomyces  from  cocoa  do  not  ferment  saccharose  as  does  Zygo- 
saccharomyces soja,  but  both  species  ferment  dextrine  strongly. 

On  the  other  hand,  Saccharomyces  soja  and  Torula  "Shoju"  seem 
to  stand  in  close  relation  to  Z.  soja  yeast;  however,  it  might  be 
appropriate  to  group  these  three  yeasts  together  into  one  and  the  same 
species.  Be  that  as  it  may,  we  will  give  it  the  name  of  Zygosaccharo- 
soja. 


260  FAMILY  OF  SACCHAROMYCETACEAE 

SACCHAROMYCES    LINDNERI.    Guilliermond 1 

This  yeast  was  isolated  from  a  ginger  alcoholic  drink  which  is 
quite  similar  to  the  English  ginger  beer.  On  beer  wort,  at  25°  C.,  the 
yeast  develops  at  the  bottom  of  the  flask  in  the  form  of  a  white  sedi- 
ment. The  cells  are  oval  or  ovoid,  rarely  round,  like  those  of  Sac- 
charomyces  ellipsoideus.  The  yeast  then  belongs  to  the  ellipsMeus 
type.  The  cells  have  an  average  dimension  of  5.2  /z  in  length  and 
4.5  IJL  in  width.  After  three  months  the  cells  in  the  sediment  take  on  a 
peculiar  appearance.  The  growth  is  vigorous  and  includes  a  large  num- 
ber of  giant  cells,  round  or  elongated,  often  in  the  shape  of  a  curl. 
In  old  cultures,  the  cells  tend  to  take  on  the  round  shape.  The  tem- 
perature limits  for  budding  on  beer  wort  are:  minimum,  below  5°  C., 
maximum,  40-41°  C.  Near  the  temperature  limits,  the  cells  have  the 
same  shape  as  at  other  temperatures.  On  beer  wort  at  25°  C.,  this 
yeast  forms  a  feeble  ring  after  12  days  but  never  produces  a  scum. 
Sporulation  is  accomplished  easily  on  slices  of  carrot,  Gorodkowa's 
gelatin  medium  and  plaster  blocks.  When  cultivated  for  a  long 
time  on  agar  it  loses  slowly  its  sporogenic  functions  as  happens  in  many 
other  yeasts  (Lindner).  The  temperature  limits  for  sporulation  have 
not  been  given  careful  study.  The  spores  are  to  the  number  of  from 
1  to  4  per  asc.  They  are  spherical  and  have  a  diameter  of  2  to  3  JJL. 
Germination  is  accomplished  exactly  as  in  Saccharomyces  chevalieri 
and  Mangini.  It  is  generally  preceded  by  a  copulation  of  spores.  On 
agar  streaks  at  25°  C.,  there  is  produced  a  grayish  white  growth.  After 
15  days  and  up  to  two  months,  the  colony  has  the  appearance  of  a 
damp  white  layer.  The  center  is  a  little  raised  and  includes  a  number 
of  marked  raised  portions.  The  periphery  is  transparent  and  is  charac- 
terized by  a  number  of  jutting-out  canals.  The  edge  is  undulated. 
Stab  cultures  in  wort  agar  give  a  funnel  shaped  growth  after  25°  C. 
The  giant  colony  on  wort  agar  at  25°  C.,  after  15  days,  is  well  de- 
veloped with  a  white,  slightly  yellowish,  color. 

This  yeast  causes  an  active  fermentation  in  beer  wort.  It  ferments 
saccharose,  levulose,  and  d-mannose,  and  dextrose  a  little,  but  has  no 
action  on  d-galactose,  lactose,  dextrine  and  maltose. 

SACCHAROMYCES  PARADOXUS.    Batschniskaia 

This  species  was  isolated  from  the  mucous  secretions  of  trees 
at  Petrograd.  The  cells  measure  3.6-7.2  X  2.6-6  ju.  They  possess 
a  rather  special  form  of  development,  the  interpretation  of  which  is 

1  Guilliermond,  A.  Monographic  de  levures  rapportees  d'Afrique  occidentale 
par  la  Mission  Chevalier.  Annales  des  Sciences  Naturelles  Botaniques,  19,  1914. 


SACCHAROMYCES  MANGINI 


261 


<o>  * 


Fig.  121-F.  —  Saccharomyces  paradoxus. 

a  and  6,  Ascs  ;  c,  d,  e,  and  /,  Fusion  of  Cells  and  Forma- 
tion of  Promycelium;  m,  g,  I,  yeast  Cells  Derived 
from  Budding  of  Copulated  Cells  Formed  by  the 
Promycelium  ;  i,  Promycelium  Transforming  Directly 
into  an  Asc  (after  Batschinskaia)  . 


difficult.  From  1  to  8  ascospores  are  formed  in  each  asc,  generally  4. 
Germination  is  preceded  by  a  fusion  of  the  ascospores.  This  is  ac- 
complished between  two  ascospores  but  usually  one  may  see  from  one 
to  three  or  a  greater  number  fusing.  The  cells  which  result  from  this 
fusion  finally  elongate  and  take 
on  various  shapes,  giving  a  sort 
of  promycelium.  In  this  pro- 
mycelium,  there  are  formed 
many  generations  of  buds.  The 
cells  which  result  from  the  bud- 
ding fuse  two  by  two,  and  these 
are  the  cells,  which  by  budding, 
produce  vegetative  cells. 
Streaks  on  gelatin  give  colonies 
which  are  white,  small  and 
striking.  On  must  agar,  the 
yeast  develops  under  the  form 
of  a  brilliant  coating,  viscous 
and  light  yellow.  The  giant 
colonies  on  must  agar  have  the 
same  color  as  the  colonies  on 
streak  cultures.  Bouillon  cultures  with  beer  wort  added  give  no  scum 
but  a  brown  sediment  and  a  cloudiness.  This  yeast  ferments  glucose, 
levulose,  saccharose  and  galactose.  A  cytological  study  of  the  yeast 
seems  advisable  in  order  to  interpret  the  cell  structure  during  the 
different  stages  of  growth. 

SACCHAROMYCES  MANGINI.    Guilliermond l 

This  yeast  was  isolated  from  fermenting  wine  Bili  made  at  Conakry 
and  was  found  along  with  a  species  named  Zygosaccharomyces  Cheva- 
lieri.  This  wine  is  a  drink  prepared  from  tubercles  of  the  Osbeckia 
grandiflora. 

On  beer  wort  at  25°  C.,  S.  Mangini  forms  an  abundant  white  sedi- 
ment. When  -examined  microscopically  after  24  hours  the  sediment 
seems  to  be  made  up  of  oval  or  round  cells  resembling  somewhat 
those  of  S.  cllipsoideus.  This  yeast  then  also  belongs  to  the  ellipsoi- 
deus  type.  The  cells  are  isolated  and  sometimes  united  into  budding 
colonies  of  from  2-4  cells.  They  are  smaller  than  the  cells  of  S. 
Chevalieri.  The  average  dimensions  are  about  4.4  ju,  wide  and  6.75  n 
in  length.  The  cells  keep  the  same  form  after  15  days. 

1  Guilliermond,  A.  Monographic  des  levures  rapporte*es  d'Afrique  occidentale 
par  la  Mission  Chevalier.  Annales  des  Sciences  Naturelles  Botaniques,  19, 
1914. 


262  FAMILY   OF  SACCHAROMYCETACEAE 

The  temperature  limits  for  budding  on  beer  wort  are  a  minimum 
below  5°  and  a  maximum  of  40^11°.  The  shape  of  the  cell  is  the  same 
at  the  temperature  limits  as  at  the  optimum  temperature.  A  feeble 
ring  but  no  scum  is  formed  at  the  end  of  11  days. 

Sporulation  is  easily  accomplished  on  slices  of  carrot  and  Gorod- 
kowa's  gelatin  and  the  plaster  block.  The  ascs  contain  from  1-4 

spherical  spores,  2-2.5  jit  in  diameter. 
Spores  germinate  exactly  as  those  of  S. 
Chevalieri.  On  wort  agar  at  25°  the  yeast 
develops  after  three  or  four  days  with  a 
train  of  white,  moist  colonies.  The  center 
is  slightly  indented  and  the  border  slightly 
sinuous.  The  center  is  thick  and  granular, 
Fig.  121-G.  —  Saccharomyces  the  periphery  surrounded  by  a  number  of 

canals  running  out  from  the  center. 

On  wort  agar  stab  cultures  at  the  end 


Germination  of  Ascospores. 

shaped.  On  wort  gelatin  at  20°  the  colony  is  round  with  a  slightly 
raised  center.  There  is  no  liquefaction  of  gelatin.  The  yeast  has  the 
characteristics  of  a  top  yeast  and  causes  a  rather  active  fermentation 
of  beer  wort.  It  ferments  saccharose,  dextrose,  levulose,  d-mannose, 
lactose,  d-galactose,  and  dextrine. 

SACCHAROMYCES   CHEVALIERI.     Guilliermond l 

This  yeast  was  isolated  in  a  wine  fermentation  from  the  Ivory 
Coast.  On  beer  wort  at  25°,  this  yeast  forms  an  abundant  white 
sediment.  When  examined  at  the  end  of  24  hours,  this  sediment  shows 
large  cells,  spherical  or  oval  in  shape.  Many  give  birth  to  long 
buds  sometimes  in  the  form  of  sausages.  A  certain  number  of  the 
cells  are  elongated,  but  the  round  or  oval  cells  are  the  most  frequent. 
The  cells  of  this  yeast  belong  to  the  ellipsoideus  type. 

The  dimensions  of  the  cells  vary  between  5  and  9  fJL  in  length  and 
4  and  7  JJL  in  width.  The  average  dimensions  are  about  5.53  /z  long  and 
4.14  IJL  wide.  The  cells  are  frequently  united  in  small  colonies  of  about 
3-10  budding  units.  In  older  cells  these  colonies  are  generally  spheri- 
cal or  oval,  while  the  young  cells  have  a  tendency  to  elongate. 

The  temperature  limits  for  budding  on  beer  wort  are  a  minimum 
below  5°  C.  and  a  maximum  of  40-41°  C.  Near  these  temperature 
limits  the  yeast  has  the  same  cellular  forms  as  at  other  temperatures. 

1  Guilliermond,  A.  Monographic  des  levures  rapportees  d'Afrique  occidentale 
par  la  Mission  Chevalier.  Annales  des  Sciences  Naturelles  Botaniques,  19, 
1914. 


SACCHAROMYCES  ETIENNE  263 

A  feeble  ring  is  formed  at  25-30°  on  beer  wort  after  about  12  days. 
This  ring  is  made  up  of  spherical  or  oval  cells  united  in  groups.  The 
mycelial  formation  has  not  been  observed. 

Spores  are  formed  quickly  on  slices  of  carrot,  Gorodkowa's  medium 
and  the  plaster  block.  The  temperature  limits  on  plaster  block  are 
maximum  39-40°  and  minimum  8-10°.  The  optimum  is  situated  at 
about  25-30°.  At  this  temperature  the  spores  appear  in  about  12 
hours.  The  spores  are  to  a  number  of  from  1  to  4  per  asc.  They 
are  spherical  and  have  a  diameter  of  from  2.5  to  3.5  microns. 

Germination  is  generally  preceded  by  sexual  processes  analogous 
to  those  which  have  been  described  for  Johannesburg  II  yeast.  At 
the  beginning  of  germination  the  spores  enlarge;  the  wall  of  the  asc 
disappears,  but  may  persist  during  the  first  stages  of  germination. 
About  one-fourth  of  the  spores  germinate  only  by  ordinary  budding 
without  preliminary  copulation.  The  others  unite  two  by  two  by 
means  of  a  copulation  canal. 

On  wort  agar  in  streaks  8.  Chevalieri  produces  at  the  end  of  three 
days  a  train  of  grayish  white  growth  with  a  slightly  indented  border; 
at  the  end  of  15  days  to  a  month  the  colony  is  white  with  a  damp 
appearance;  its  center  is  thick  and  border  slightly  undulated.  On 
wort  gelatin  in  stab  cultures  the  yeast  develops  a  colony  which  is 
funnel  shaped.  The  surface  has  a  damp  appearance;  it  is  thick  at 
the  center  and  thin  at  the  edges.  The  giant  colony  on  wort  agar 
at  the  end  of  15  days  at  25°  is  well  developed,  spherical,  slightly 
moist,  and  has  a  grayish  white  color.  It  is  made  up  of  a  central  granu- 
lar portion  and  a  thin  transparent  peripheral  part. 

S.  Chevalieri  has  the  characteristics  of  a  top  yeast.  It  causes  a 
rather  active  fermentation  on  beer  wort.  It  ferments  saccharose, 
dextrose,  levulose  and  d-mannose  quite  actively,  but  does  not  seem 
to  have  any  action  on  galactose,  maltose  or  lactose. 

SACCHAROMYCES  ETIENNE.    Potron » 

This  yeast  was  isolated  from  sputum  from  a  disease  in  which  it 
was  the  causal  agent.  The  infection  began  with  a  gastro-enteritis 
which  later  turned  into  a  pleuro-pulmonary  trouble  which  had  some 
of,  the  appearances  of  tuberculosis.  The  sickness  yielded  to  treat- 
ment with  iodine.  The  yeast  develops  on  carrot  and  potato.  It  has 
cells  which  vary  from  spherical  to  ellipsoidal  in  shape  (3-9  /z  long 
and  4-5  jit  wide).  Curled  cells  are  more  numerous  in  scums  and  old 
cultures.  The  ascospores  appear  on  carrot  after  30  hours.  The 

1  Potron.  Presence  (Tune  levure  au  course  d'une  infection  pleuropulmonaire 
grave.  Soc.  de  Med.  de  Nancy,  1914. 


264  FAMILY  OF  SACCHAROMYCETACEAE 

ascs  usually  contain  4  elliptical  ascospores  (2-2.5 /x).  The  wall  of  the 
asc  is  broken  when  the  spores  germinate.  Ger- 
mination  is  by  ordinary  budding.  On  potato  at 
25°  C.,  punctiform  spherical  colonies  are  formed 
having  a  grayish  white  color.  These  become  con- 
fluen^  mto  large  colonies  with  festooned  edges. 
White  confluent  colonies  develop  on  carrot.  The 
yeast  ferments  glucose. 


D.     Fourth  Sub-Group 
Fig.    121-H.—  Sac- 
charvmyces  Etiennei         Yeasts  fermenting  dextrose,  but  having  no  action 

0  ron''          on  saccharose,  maltose  or  lactose. 


SACCHAROMYCES  MALI  DUCLAUXI.     Kayser 

This  species  was  found  by  Kayser  1  in  a  sample  of  cider.  The 
cells  are  large  (6  to  12  /*  long  and  4  to  7  IJL  wide)  and  form  a  light  float- 
ing growth.  They  are  killed  at  55°  C.  and  are  very  sensitive  to  acids. 
Ascospores  are  formed  at  the  end  of  30  hours  at  15°  C.  This  yeast 
acts  on  neither  saccharose  nor  maltose  but  ferments  invert  sugar  im- 
parting a  "  bouquet  "  to  the  solutions. 


SACCHAROMYCES  UNISPORUS.    Jorgensen2 

This  yeast  was  discovered  by  Holm.  By  its  action  on  sugars,  it 
is  related  to  Saccharomyces  mali  Duclauxi.  The  cells  are  small  and 
oval.  In  old  cultures,  one  may  see  the  shape  of  the  cells  of  Pasto- 
rianus.  The  spores  appear  at  the  end  of  40  hours  at  25°  C.  After 
72  hours  at  15°  C.  only  a  few  ascs  are  visible.  The  ascospores  are  round 
and  refractive.  This  yeast  does  not  form  a  scum  but  simply  a  ring 
in  old  cultures. 

E.    Fifth  Sub-Group 

Yeasts  which  ferment  lactose. 

SACCHAROMYCES  FRAGILIS.    Jorgensen3 

This  yeast  was  encountered  in  kefir,  an  alcoholic  milk  produced 
by  the  fermentation  of  S.  fragilis  and  many  Torula  and  many  bac- 
teria among  which  is  Bacillus  caucasicus.  Saccharomyces  fragilis  pos- 

1  Kayser,  E.    Etudes  sur  la  fermentation  du  cidre.     Ann.  Past.  Inst.  4,  1890. 

2  Jorgensen,  A.     Die  Mikroorganismen  der  Garungsindustrie,  Berlin,  5th  edi- 
tion, Paul  Parey,  1909. 

*  Jorgensen,  A.    See  reference  for  S.  unisporus. 


SACCHAROMYCES   ACIDI   LACTICI  265 

sesses  small  oval  or  elongated  cells.     (Fig.  122,  A.)    The  temperature 
optimum  for  budding  is  towards  30°  C.     Ascs  are  produced  on  plas 
ter  blocks  at  25°  C.  in  twenty  hours,  and  at  15°  C.  in  forty  hours.    They 
are  also  formed  in  fermenting  solutions  and  on  gelatin.     The  asco- 
spores  are  round  or  elongated  (Fig.  122,  B).     After  a  long  time,  this 


B 

Fig.  122.  —  Saccharomyces  fragilis.     A,  Young  Vegetation  on 
Yeast  Water  with  Added  Lactose.     B,  Ascs  (after  Holm). 

yeast  produces  a  scant  scum.  At  room  temperature  this  yeast  forms 
about  1  per  cent  of  alcohol  by  volume  after  eight  days  and  4  per 
cent  after  4  months.  In  beer  wort,  after  six  days,  about  1  per  cent 
of  alcohol  is  formed.  According  to  Bau  this  yeast  ferments  lactose, 
but  has  no  action  on  melibiose. 


SACCHAROMYCES  FLAVA  LACTIS.    Krueger l 

This  species  is  found  in  beer  to  which  it  contributes  an  abnormal 
yellow  color,  and  a  disagreeable  odor,  resembling  putrefied  urine.  It 
possesses  small  cells  attached  in  chain  formation.  They  sporulate 
rapidly  on  slices  of  carrot.  The^  colonies  on  gelatin  are  yellow.  Gela- 
tin is  liquefied  very  rapidly  and  the  colonies  cover  themselves  with  a 
scum.  This  yeast  produces  a  yellow  scum  in  milk  and  in  a  decoc- 
tion of  milk  sugar.  The  coloring  matter  is  formed  only  in  contact 
with  air. 

SACCHAROMYCES  ACIDI  LACTICI.     Grotenfelt 

Grotenfelt2  has  described  under  this  name  a  yeast  which,  intro- 
duced into  sterile  milk,  causes  a  coagulation  and  at  the  same  time 
a  formation  of  acid.  Its  cells  are  ellipsoidal  (2.0-4.35  ju  long  and  1.5- 
2.9  jj,  wide).  On  gelatin,  and  on  agar,  it  forms  white  shiny  colonies. 
On  potato  it  forms  large  moist  spots  clear  gray  which  turn  to  a  brown. 

1  Krueger,   R.     Bakter.    chemische   Untersuchungen   kasiger   Butter.     Cent. 
Bakt.  7,  1890. 

2  Grotenfelt,    G.     Ueber   die   Spaltung   von   Milchzucker   durch   Sprosspilze 
und  iiber  schwarzen  Kase.    Fortschr.  der  Med.  7,  1889. 


266  FAMILY  OF  SACCHAROMYCETACEAE 

It  ferments  lactose  giving  0.108  per  cent  of  alcohol  in  eight  days. 
Grotenfelt  pretends  to  have  obtained  ascospores  on  potato  but  the 
existence  of  these  ascospores  does  not  seem  to  be  well  established. 
Then,  again,  it  is  possible  that  this  species  may  be  a  Torula. 


SACCHAROMYCES  LACTIS  a.    Dombrowski l 

This  species,  isolated  from  Bulgarian  yoghourt,  has  been  described 
in  the  laboratory  of  Professor  Jensen  at  Copenhagen  by  Dombrowski. 
Generally  it  is  a  yeast  with  elliptical  cells  but  it  may  present  cells 
elongated  to  18  JJL  especially  on  solid  media.  On  grape  must,  the  cells 
may  be  9.0-6.0  /*  long  and  3.75-3.25-3.00  ju  wide.  In  cultures  on  the 
cover  glass  the  formation  of  giant  colonies  is  easily  observed. 

Sporulation  is  difficult  to  obtain  especially  after  successive  cultur- 
ing  in  liquid  media.  Ascospores  are  formed  at  the  end  of  about  44  hours 
at  25°  C.  in  cells  arising  from  solid  media.  The  ascospores  are  round 
and  to  the  number  of  three  to  four  in  each  asc.  They  germinate  by 
ordinary  budding. 

On  beer  must  gelatin  in  plates,  the  colonies  are  lenticular.  In 
stabs,  the  growth  is  along  the  line  of  inoculation  as  one  approaches 
the  surface.  Giant  colonies  on  beer  must  gelatin,  after  two  months, 
offer  a  delicate  structure  with  concentric  zones  and  rays  running  out 
from  the  center. 

Saccharomyces  lactis  a  acts  like  a  bottom  yeast  in  sugar  solutions. 
It  never  produces  a  scum,  but  a  ring  is  formed  at  the  end  of  six  weeks 
at  room  temperature.  In  beer  must,  it  produces  a  fermentation  ac- 
companied by  the  formation  of  an  agreeable  odor.  It  decolorizes 
the  wort  in  10  days  and  yields  after  4J  months  four  to  five  grams  of 
alcohol  per  100  c.c.  It  causes  an  active  fermentation  in  milk  at  23- 
25°  C.  It  ferments  lactose,  saccharose  and  dextrose  but  does  not  act 
on  maltose.  A  small  amount  of  acids  are  found  among  the  products 
of  the  fermentation. 

SACCHAROMYCES  LACTIS  ft.     Dombrowski1 

This  species  has  been  isolated  by  Dombrowski  from  a  sample  of 
milk  fermented  at  45°  C.  The  cells  possess  variable  forms.  One  may 
find  egg-shaped  or  elliptical  cells  aside  from  the  elongated  cells  which 
may  reach  20  microns.  These  latter  cells  remain  attached  and  form 
long  chains  or  a  sort  of  mycelium.  On  beer  wort,  the  cells  measure 
7.6-5.5-4.6-3.8  JLC  in  length  and  4.6-3.8  ju  in  width.  Sporulation  is  ac- 

1  Dombrowski,  W.  Die  Hefen  in  Milch  und  Milchprodukten.  Cent.  Bakt. 
28,  1909. 


YEAST   FROM   KOUMYS  267 

complished  easily.  They  appear  at  the  end  of  five  hours  at  25°  C.  on 
plaster  blocks.  They  may  also  be  observed  in  cultures  on  gelatin,  in 
hanging  drops  and  in  the  scums  of  old  cultures  on  liquid  media.  The 
ascospores  are  to  the  number  of  from  one  to  eight  per  asc.  Their 
form  and  dimensions  are  variable.  More  often  they  are  elliptical 
but  sometimes  they  are  hemispherical.  They  germinate  by  ordinary 
budding  after  absorbing  the  wall  of  the  asc.  The  colonies  on  nutrient 
agar,  in  plates,  are  circular  and  torpedo  shaped.  In  stabs,  the  growth 
is  along  the  line  of  inoculation  and  increases  towards  the  surface. 
Giant  colonies  show  a  center  with  a  crateriform  concavity  with  radii 
out  from  the  center.  Saccharomyces  lactis  ft  produces  in  liquid  media 
with  sugars,  a  scum  and  a  ring  in  which  the  cells  have  somewhat  the 
form  of  a  mycelium  with  ascospores.  It  acts  as  a  bottom  yeast.  An 
active  fermentation  is  produced  during  which  a  feeble  aroma  may  be 
noticed.  Beer  wort  is  distinctly  decolorized  and  there  is  formed  at  the 
end  of  five  and  a  half  months  7.93  per  cent  of  alcohol  by  volume.  In 
milk  an  energetic  fermentation  is  produced  at  23-25°  C.  It  ferments 
saccharose,  lactose,  d-galactose  and  dextrose  but  has  no  action  on 
maltose.  It  seems  to  be  closely  related  to  Saccharomyces  fragilis 
(Jorgensen). 

YEAST  FROM  KOUMYS.     Schipin 

This  yeast  was  isolated  from  koumys  by  Schipin.  Along  with  a 
few  bacteria,  it  contributes  to  the  formation  of  koumys  by  inducing 
a  fermentation  in  milk  with  a  small  quantity  of  lactic  acid.  Ru- 
binsky  l  has  given  a  detailed  description  of  this  organism.  In  hanging 
drops  this  yeast  possesses  round  or  oval  forms  which  contain  at  one 
of  their  extremities  or  in  the  middle  a  large  refractive  granule.  In 
old  cultures  on  agar,  in  Petri  dishes  (after  four  or  five  weeks)  the 
cells  are  often  elongated.  Old  cells  always  contain  granules. 

Schipin  has  obtained  sporulation  in  this  yeast.  Rubinsky  has  also 
observed  on  plaster  blocks  the  formation  of  six  or  eight  globules  which 
look  like  ascospores  but  it  is  not  certain  that  they  are  true  ascospores. 
On  gelatin  plates,  this  koumys  yeast  exhibits  mediocre  development 
during  the  first  few  days.  At  the  end  of  three  days,  it  forms  surface 
colonies  of  about  2-3.5  millimeters  and  the  deep  colonies  are  round. 
The  culture  gives  off  an  aromatic  odor  often  acid.  On  gelatin  stabs, 
the  culture  takes  the  form  of  a  bottle  and  has  a  flat  white  color  some- 
times yellow  along  the  line  of  puncture.  On  gelatin  added  to  bouillon, 
after  2  to  3  weeks,  the  colonies  have  a  center  sometimes  soft  and  shiny. 
In  cabbage  bouillon,  the  colonies  have  a  peculiar  appearance.  The 
1  Rubinsky,  B.  Studien  iiber  den  Koumiss.  Cent.  Bakt.  28,  1910. 


268  FAMILY  OF  SACCHAROMYCETACEAE 

middle  is  thin,  dull,  finely  granular  and  dry.  The  edge  is  distinctly 
raised  above  the  central  part  surrounded  by  a  granular  portion. 

The  colonies  on  gelatin  possess  no  characteristic  appearance.  The 
yeast  gives  a  growth  resembling  a  string  of  pearls.  The  cultures  on 
gelatin  after  from  3  to  5  weeks  show  a  liquefaction.  At  37°  the  cul- 
ture is  juicy,  shiny  and  white.  It  forms  great  white  colonies.  Later 
they  spread  all  over  the  surface. 

In  milk  at  37°  C.  the  koumys  yeast  produces  a  strong  fermentation 
of  the  lactose  and  yields  about  36  per  cent  of  lactic  acid.  The  liquid 
is  cloudy  at  first  but  clears  up  showing  an  abundant  vegetation  in 
the  sediment  of  a  varying  white  or  yellow  color.  It  never  produces  a 
scum.  It  has  the  characteristics  of  a  bottom  yeast.  It  decomposes 
the  casein  which  it  changes  to  albumoses  and  peptones  and  produces 
aromatic  ethereal  substances  which  impart  to  koumys  its  aroma. 

SACCHAROMYCES  ANAMENSIS.    Will1 

This  yeast,  which  has  been  employed  in  distilleries  under  the 
name  of  "Levure  anamite,"  is  a  top  yeast  of  the  wild  variety. 
The  cells  are  generally  oval,  sometimes  elongated  (4  to  9  ju).  Giant 
cells  may  be  noticed  in  cultures.  Groups  are  rarely  formed  by  indi- 
vidual cells.  The  spherical  ascospores,  one  to  four  per  asc  (2.4  to  4  /z) 
have  an  optimum  temperature  for  sporulation  of  33°  C. ;  a  maximum 
of  35°  C.  and  a  minimum  of  12°  C.  This  yeast  forms  a  scum  made  up 
of  round  or  oval  cells,  sometimes  with  cells  shaped  like  a  sausage. 
The  optimum  temperature  for  the  formation  of  scums  on  beer  wort  is 
near  31°  C.  This  yeast  ferments  dextrose,  levulose,  galactose,  saccha- 
rose, maltose  and  raffinose.  Lactose  is  assimilated,  but  the  yeast  is 
able  to  ferment  it  but  feebly. 

SACCHAROMYCES  TAETTE,  Major  and  Minor 
Olsen-Sopp 2 

These  two  yeasts  were  isolated  from  Taette,  a  milk  used  in  times 
of  antiquity  by  the  people  of  the  North  (Norway  and  Sweden). 
It  is  a  thick  viscous  milk,  not  coagulated,  but  with  an  acid  taste 
which  is  quite  agreeable.  Saccharomyces  taette  major  is  distinguished 
from  the  second  variety  by  the  fact  that  its  cells  are  much  larger  and 
that  it  produces  ascospores.  The  second  type,  Saccharomyces  taette 
minor,  does  not  give  ascospores.  Taette  does  not  contain  over  0.3 

1  Will,  H.     Saccharomyces  anamensis,  die  Hefe  des  neuen  Amyloverfahrens. 
Cent.  Bakt.  Abt.  2,  39  (1913)  26. 

2  Olsen-Sopp,  O.  J.     Taette,  the  primitive  Norse  storage  milk  and  associated 
milks;   their  significance  as  a  nutrient.    Cent.  Bakt.  Abt.  2,  33  (1912),  1-5. 


SACCHAROMYCES  THEOBROMAE  269 

to  0.5  per  cent  of  alcohol  by  volume  which  results  from  a  fermen- 
tation of  the  lactose  by  these  two  species. 

F.     Sixth  Sub-Group 

Yeasts  which  do  not  produce  alcohol  and  in  which  the  character- 
istics of  fermentation  are  little  known. 


SACCHAROMYCES   CONGLOMERATUS.     Reess 

This  yeast  was  found  by  Reess  l  on  decayed  grapes  and  at  the 
beginning  of  the  wine  fermentation.  The  description  of  the  author  is 
reproduced  here.  "Budding  cells,  round,  5  to  6  ju  in  diameter  united 
in  bunches.  This  formation  is  accomplished  in  the  following  man- 
ner. On  the  axis  of  two  old  cells,  before  they  germinate,  by  budding 
in  the  direction  of  their  longitudinal  axis  there  are  formed  simul- 
taneously many  buds  which  branch  out.  The  ascs  are  frequently 
united  two  by  two  in  each  cell.  Two  to  four  spores  are  present  which 
during  germination  give  rise  to  new  bunches." 

Hansen  has  never  observed  a  yeast  which  may  be  compared 
to  Saccharomyces  conglomeratus.  He  thinks  that  this  yeast  may 
not  be  a  well-differentiated  yeast  but  may  be  a  yeast  which  has 
been  studied  under  another  name  (S.  pastorianus,  ellipsoideus,  etc.). 
These,  in  their  scums,  present  often  the  appearance  described  by 
Reess  in  S.  conglomeratus.  The  existence  of  this  yeast  is,  then,  prob- 
lematical. 

SACCHAROMYCES  HANSENII.     Zopf2 

This  yeast  has  been  isolated  from  the  pollen  of  cotton.  The  cells 
are  spherical  or  ellipsoidal  and  each  one  contains  many  fat  globules. 
They  measure  4  to  11  JJL.  The  ascospores  are  spherical  and  to  the 
number  of  two  per  asc.  The  cultures  on  must  gelatin  in  stabs  are 
a  brilliant  white  with  no  liquefaction.  In  solution  of  dextrose,  d- 
galactose,  lactose,  maltose,  dulcite,  glycerol  and  mannite,  this  yeast 
produces  varying  amounts  of  oxalic  acid. 

SACCHAROMYCES  THEOBROMAE.     Preyer3 

This  yeast  completes  the  fermentation  of  cocoa.  In  scums,  its 
cells  are  long,  cylindrical  rods.  In  culture  solutions  when  poorly 

1  Reess,  M.     Ueber  den  Soorpliz  Sizungsber.  der  physikalisch.  rnbd.  Sozietat. 
Erlangen  Botan.  Ztg.  1878. 

2  Zopf,  W.     Oxalsauregarung  bei  einem  typischen  Saccharomyceten  (S.  Han- 
senii).    Ber.  Bot.  Gesell.  7,  1889. 

3  Preyer,  A.    Der  Tropenflanzer,  1901. 


270  FAMILY  OF  SACCHAROMYCETACEAE 

nourished,  this  species  produces  ascospores  after  18  hours  at  25°  C. 
They  are  very  numerous  in  each  asc.  In  decoctions  of  cocoa  this 
yeast  produces  an  alcoholic  fermentation  and  forms  a  scum.  It  does 
not  invert  saccharose  and  dies  rapidly  in  this  solution. 

YEAST  FROM   SALT!     Hoye 1 

This  species  was  found  by  Hoye  in  the  analysis  of  air.  This 
author  used  as  a  nutrient  medium  a  wheat  paste  to  which  was  added 
17  per  cent  of  salt.  It  is  a  round  yeast  which  forms  a  single  spore 
in.  each  asc.  The  best  medium  is  a  fish  bouillon  with  10  per  cent  of 
salt  added.  In  nutrient  liquids  with  3  per  cent  of  salt,  development 
ceases  completely.  The  different  proportions  of  salt  have  no  influence 
on  the  shape  of  the  cells.  This  yeast  produces  no  fermentation  in 
apple  juice. 

SACCHAROMYCES  ANGINAE.    Achalme  and  Troisier2 

This  species  was  found  by  Achalme  and  Troisier  in  a  clinical 
angina  analogous  to  thrush.  The  cells  are  oval  (8  to  9  by  5-6  ju), 
isolated  in  groups  of  8  to  10,  often  budding  at  one 
of  the  poles.  (Fig.  123.)  In  cultures,  they  pro- 
duce ascs  with  4  rounded  ascospores  (2  microns). 
On  gelatin,  this  yeast  develops  with  grayish  white 
colonies,  with  the  deep  colonies  brown  and 
Fig.  123.  —  Saccharo-  spherical.  S.  anginae  does  not  liquefy  the  gelatin. 

mycesanginae.  Bud-  Qn  agar  the  colonies  are  thick  and  of  a  dull  rose, 
ding  Cells  and  Ascs  .  ,  ,     .        ,       , 

(after  Troisier  and   In   acidulated   water,   the  growth  is   cloudy  and 
Achalme).  tnere   js  produced   at   the   end   of  three  days  a 

sticky  brown  deposit.     This  yeast  ferments  saccharose. 

SACCHAROMYCES  TUMEFACIENS.     (Curtis)  Busse 
Syn.:   SACCHAROMYCES  SUBCUTANEOUS  TUMEFACIENS.     Curtis3 

This  species  was  observed  by  Curtis  from  a  tumor  of  the  hip 
and  from  a  lumbar  abscess  in  man.  In  the  tumor  it  presents  oval  or 
spherical  cells  with  granular  contents  (16  to  20  ju)  and  is  surrounded 
by  a  gelatinous  capsule  (Fig.  124.)  In  cultures,  the  cells  oval  at  first 

1  Hoye,  K.    Rech.  sur  la  moisissure  de  Bacalao  et  quelques  autres  microorga- 
nismes  halophiles.    Bergens  Museums  Aarbog.  No.  12,  1906. 

2  Achalme  and  Troisier.     Sur  une  angine  parasitaire.     Arch.   med.  Exp.  3, 
1895. 

3  Curtis,   F.     Contribution  a  Petude  de  la  Saccharomycose  humaine.     Ann. 
Past.  Inst.  10,  1895. 


SACCHAROMYCES  BLANCHARDII  271 

without  capsules  sometimes  form  in  chains  of  two  or  three.     The  cap- 

sule may  develop  in  old  cultures.    This  yeast  often  forms  ascs  with 

from  1  to  4  ascospores  (Busse).     In  gelatin  stabs, 

a  white  growth  is  secured  after  48  hours.     The 

gelatin  is  not  liquefied.     On  agar  the  colonies 

are  small,  and  lunctiform  and  fuse  quickly  into 

a   solid  mass.    On  glycerol   potato  the   growth 

is  rapid.     The  culture  has  the  appearance  of  a 

continuous  dry  streak,  at  first  white  and  later 

brown.     On  liquid  media,  beer  wort,  the  develop- 

ment   is    rapid    and    abundant.      No    scum    is  Fig.  124.  —  Saccharorhy- 

formed.     This    yeast    inverts    saccharose,    and 


causes  a  feeble  fermentation.     Alcohol  and  acid      with  Gelatinous  Cap- 
are  formed.     There  is  no  action  on  maltose  nor 

lactose.      Its   maximum  temperature   is   around    37°  C.     This  yeast 
has  a  local  pathenogenicity  for  the  rat,  the  white  mouse,  and  the  dog. 

SACCHAROMYCES  GRANULATUS.    Vuillemin  and  Legrain 

This  yeast  was  isolated  by  Vuillemin  and  Legrain.1  It  possesses 
oval  or  elliptical  cells,  sometimes  globular  or  elongated,  about  2- 
10  jit  X  2-4  fj,.  The  membrane  is  covered  with  granulations  which  are 
either  irregular  or  regular.  (Fig.  125.)  These 
cells  form  one,  rarely  two  or  three  buds,  and  con- 
tain fat  globules  of  a  reddish  color.  The  cultures 
thus  take  on  a  vermillion  tint.  Some  of  the  cells 
are  able  to  encyst  and  change  into  durable  cells 
or  chlamydospores.  On  plaster  blocks,  the  cells 
have  very  thin  and  folded  membranes  containing 
g  125.  _  Sarcharo-  two  or  f°ur  ascospores  which  are  spherical  or 
myces  granulatus.  elliptical.  On  liquid  media  this  yeast  does  not 
Pr°duce  a  scum  but  a  sediment  is  formed  which 
is  reddish  in  color.  In  gelatin  stabs,  punctiform 
colonies  are  formed.  The  gelatin  is  not  liquefied.  On  agar,  beet, 
carrot,  or  cabbage,  it  forms  a  folded  and  shiny  coating.  On  potato 
slants,  the  growth  is  dry.  This  yeast  is  pathenogenic  for  the  rabbit. 

SACCHAROMYCES  BLANCHARDII.     (Blanchard).   Guiart  2 

This  yeast  described  by  Blanchard,3  Schwartz  and  Binot  was  iso- 
lated from  a  tumor  of  a  man.    It  was  taken  from  the  peritoneum  after 

1  Vuillemin,  P.,  and  Legrain.    Sur  un  cas  de  Saccharomycose  humaine.    Arch. 
de  Parasit.,  3,  1900. 

2  Guiart,  J.     Precis  de  parasite-logic.     Bailliere,  1910. 

3  Blanchard,   R.,  Schwartz,  E.,  and  Binot   J.     Sur  une  Blastomycose  intra- 
peritoneale.     Arch,  de  Parasit.,  7,  1903. 


272 


FAMILY  OF  SACCHAROMYCETACEAE 


which  the  patient  succumbed  to  the  operation.  It  has  round  cells, 
(1.5  to  15  or  20  p)  slightly  greenish,  surrounded  by  a  thick  mucilag- 
inous capsule.  In  cultures  on  carbo- 
hydrate agar,  the  cells  are  also  spheri- 
cal and  often  appear  in  beaded  forma- 
tion. (Fig.  126.)  Almost  all  of  them 
after  a  few  weeks  transform  into 
spherical  ascs  with  a  thick  wall.  They 
contain  8  round  ascospores.  These 
measure  34  /x  in  diameter.  This  species 
grows  on  liquid  media  and  produces 
a  sediment.  On  gelatin  streaks,  it 
gives  a  grayish  white  colony.  The 

On  gelatin 

plates,  white  colonies  are  produced 
which  are  round  or  scalloped  and  on 
agar  a  thick  growth,  a  little  yellow  and  not  scalloped.  On  agar 
plates,  it  forms  lenticular  spots  yellow  or  grayish.  On  potato,  a 
mucous  coating,  white  or  yellowish,  is  formed  and  on  carrot  an 
abundant  viscous  growth. 


Fig.    126.  —  Saccharomyces    Blan- 

chardi.      Sucrose    Agar    Culture    gelatin  IS  slowly  liquefied, 
after  48  Hours  (after  Blanchard, 
Schwartz,  and  Binot). 


SACCHAROMYCES  MINOR.     Engel1 

The  yeast  was  found  in  a  fermentation  of  bread.  It  has  spherical 
cells  6  ju  in  diameter,  united  in  chain  formation  and  in  little  groups 
of  six  or  nine  cells.  The  ascs  are  7.8  ju,  and  contain  2  to  4  ascospores 
of  3  ju  in  diameter. 


SACCHAROMYCES  UVARUM.    Beijerinck2 

This  yeast  is  not  well  known.  It  was  found  in  a  bottle  of  grape 
juice  to  which  saccharose  had  been  added.  It  produced  an  active  fer- 
mentation and  was  associated  with  S.  sphaericus  (Naegeli).  It  is  a 
yeast  which  especially  ferments  maltose.  In  yeast  water  to  which 
maltose  is  added  it  produces  acetic  acid.  On  nutrient  gelatin,  ascs 
are  formed  easily  and  in  large  numbers  with  4  ascospores. 

1  Engel,  Les  ferments  alcooliques.     Thesis  for  the  Doctorate  of  Sciences,  Paris, 
1872. 

2  Beijerinck,  M.  W.    Ueber  Regeneration  der  Sporenbildung  bei  Alkoholhefen. 
Cent.  Bakt.  4,  1898. 


HANSENIA  APICULATA  273 


SACCHAROMYCES  LEMONNIERI.    Sartory  and  Lasseur  l 

Isolated  from  the  sputum  of  a  person  with  bronchitis  and  pul- 
monary congestion,  this  yeast  has  round  cells  surrounded  with  a 
thick  capsule.  It  grows  easily  on  most  media.  The  optimum  tem- 
perature for  growth  is  between  25  and  30°  C.  It  vegetates  quite  well 
at  37.5°  C.  but  stops  at  40°  C.  It  forms  a  scum  on  glycerol  bouillon. 
The  temperature  limits  for  scum  formation  are,  15-20°  C.  and  37- 
38°  C.  Asc  formation  has  been  obtained  on  plaster  blocks,  each  asc 
containing  four  spores  which  are  spherical  in  shape  (2.5-3  ju).  On 
carrot,  development  is  rapid,  a  white  thick  colony  being  obtained. 
On  potato,  the  growth  is  scant.  It  is  pathogenic  for  rabbits  and 
guinea  pigs. 

Genus  X.     Hansenia.     Lindner- Klocker 

HANSENIASPORA.     Zikes 

Cells  lemon  shaped,  with  hemispherical  ascospores,  and  provided 
with  a  more  or  less  projecting  collar  which  gives  them  the  appearance 
of  a  hat.  They  germinate  by  ordinary  budding. 

HANSENIA  APICULATA.     Lindner2 
Syn.:  HANSENIOSPORA  APICULATA.    Zikes 

Reess  has  designated  under  the  name  Saccharomyces  apiculatus  a 
yeast  of  peculiar  shape  characterized  by  the  presence  at  one  or  both 
ends  of  an  oval  cell,  of  a  little  point  (nipple)  rather  long  which  causes 
the  cell  to  have  an  apiculate  shape.  Hansen  3  has  found  a  yeast  which 
seems  to  correspond  with  that  of  Reess'  to  which  he  gave  the  same 
name.  This  species  is  found  in  abundance  on  sugary  fruits,  in  the 
mucous  secretions  of  trees,  the  nectar  of  flowers,  etc.  It  is  encountered 
in  the  first  phases  of  the  wine  fermentation. 

The  cells  are  apiculate  and  generally  have  apiculate  buds.  Oval 
cells  with  oval  buds  may  also  be  obtained  (Fig.  6).  Under  certain 

1  Sartory,  A.,  and  Lasseur,  P.     New  pathogenic  yeast  (Saccharomyces  lemon- 
nieri.     Comp.  Rend.  Soc.  Biol.  78  (1915),  48-49. 

2  Lindner,    P.     Sporenbildung  bei   S.    apiculatus.     Woch.   f.   Brau.   No.   42, 
1903;     Mikroskopische    Betriebskontrolle    in    den    Garungswerben,    Paul    Parey, 
Berlin,  6th  edition,  1909. 

3  Hansen,  E.  C.     Recherches  sur  la  physiologic  et  la  morphologic  des  fer- 
ments alcooliques.     Sur  le  S.  apiculatus  et  sa  circulation  dans  la  nature.     Comp. 
Rend,  du  labor,  de  Carlsberg,  Vol.  1,  Book  4,  1881. 


274  FAMILY  OF  SACCHAROMYCETACEAE 

circumstances  the  cells  may  be  half-moon  shaped  or  elongated.  The 
buds,  in  the  form  of  a  lemon,  develop  especially  in  the  first  phases 
of  the  culture  and  later  may  be  replaced  by  buds  oval  in  shape.  The 
cells  never  contain  glycogen. 

Engel  has  reported  the  observation  in  this  yeast  of  ascogenous 
fructification  related  to  that  of  Protomyces  and  has  created  the  genus 
Carpozyma  for  it.  On  the  contrary,  Hansen  has  never  noticed  ascs 
or  other  forms  of  fructification  in  this  yeast  and  regards  it  as  be- 
longing to  the  Torula.  However,  Beijerinck  (1894) 
reported  the  presence  of  ascs  with  many  ascospores. 
Klocker,  however,  has  been  unable  to  confirm  the 
presence  of  ascs  and  thinks  that  Beijerinck  has  taken 
for  ascospores  the  fat  globules  which  are  commonly 
present  in  the  cells  of  this  yeast. 

Lindner  has  demonstrated  the   formation   of  ascs 
^n       Hansenia  ^n  a  Saccharomyces  apiculatus  isolated  from  the  flowers 
apiculata  (after  of  Robinia  pseudoacacia.     The  ascs  never  contain  but 
a  single  ascospore.     (Fig.  127.)     He  was  not  successful 
in  observing  the  germination  of  these  ascospores.     Rohling  has  been 
able  to  follow  the  germination  of  one  of  these  ascospores  in  a  decoc- 
tion of  horse  manure  to  which  5  per  cent  of  glucose  was  added.     It  is 
then  probable  that  special  conditions  are  necessary  for  their  germina- 
tion. 

In  the  light  of  these  discoveries,  Lindner  has  created  for  this  species 
a  new  genus  Hansenia.  But  according  to  Klocker,1  Saccharomyces 
apiculatus  in  which  Lindner  has  described  ascospores,  may  not  be 
identified  with  the  Saccharomyces  apiculatus  of  Hansen  but  may  only 
be  a  species  related  to  this  yeast,  for  in  the  true  S.  apiculatus,  under 
no  circumstances  may  the  presence  of  ascs  be  observed.  Indeed,  all 
of  the  efforts  put  forth  by  Hansen  and  Klocker  to  demonstrate  spores 
in  this  species  have  been  in  vain. 

Zikes,2  on  the  other  hand,  has  tried  to  make  Saccharomyces  api- 
culatus sporulate  by  various  procedures  but  has  had  little  success. 
He  admits  that  Hansenia  apiculata  is  different  from  S.  apiculatus 
and  proposed  to  designate  the  Saccharomyces  apiculatus  of  Hansen 
under  the  name  of  Hanseniaspora  mucroniata  (Lindner).  The  genus 
Hanseniaspora  would  be  part  of  the  family  of  Saccharomycetes  while 
the  genus  Hansenia  would  be  placed  among  the  Non-Saccharomycetes. 
It  may  be  regarded  as  an  asporogenic  variety  of  Hansenia  or  as  a  spe- 
cial form  not  having  all  of  the  characteristics  of  the  latter. 

1  Klocker,  A.     Invertin  und  Sporenbildung  bei  Saccharomyceten  apiculatus- 
Formen.    Cent.  Bakt.  21,  1910. 

2  Zikes,  H.    Zur  Nomenclaturfrage  der  Apiculatushefe.     Cent.  Bakt.  30,  1911. 


HANSENIA   VALBYENSIS 


275 


We  owe  to  Klocker  l  a  very  important  study  of  apiculate  yeasts. 
This  author  has  isolated  a  series  of  forms  made  up  of  different  species 
which  have  been  described  under  the  name  of  Saccharomyces  apicu- 
latus.  Klocker  concluded  that  this  is  not  a  special  species  but  simply 
a  group  of  species.  He  has  isolated  two  groups  of  species  which  do 
not  sporulate  and  which  he  has  incorporated  in  the  family  of  Toru- 
laceae  or  Non-Saccharomycetes  with  the  generic  name  of  Pseudosac- 
charomyces,  replacing  the  genus  of  Hansenia  of  Zikes.  The  sporulat- 
ing  species  he  has  placed  in  the  Saccharomycetes  under  the  name  of 
Hansenia  (Lindner),  replacing  the  genus  Hanseniaspora  of  Zikes. 


HANSENIA  VALBYENSIS.    Klocker 

On  beer  wort,  at  27°  C.,  the  cells  are  apiculate  or  shaped  like 
ellipsoideus;  some  are  shaped  like  short  sau- 
sages (5-8  ju).  The  limits  of  temperature 
for  growth  are  32°-33°  C.  and  0.5°  C.  The 
spores  appear  at  the  end  of  from  4  to  5  days 
on  wort  gelatin  at  25°  C.  They  are  hemi- 
spherical and  to  the  number  of  two  per  asc, 

rarely  one.   It  fer- 

ments    dextrose, 

levulose    and    d- 

mannose.  Gelatin  Fig.   128.  —  Hansenia  Val- 

is  liquefied    It  was       tyensis.     Cells     after     3 

found  at    Copen-       (after  Klocker). 
hagen. 

FOURTH   GROUP 

Budding  yeasts,  without  copulation  in 
the  origin  of  the  asc.  These  yeasts  form 
a  scum  on  sugar  media  which  is  dry  and 
opaque.  The  ascospores  are  hemispheri- 
cal in  the  form  of  a  lemon  supplied  with 
3  a  projecting  collar  and  a  single  thick  mem- 

Fig.  129.  —  Hansenia  valbyensis.  brane.  Germination  is  sometimes  pre- 
ceded by  a  parthenogamy.  The  greater 
part  of  the  species  in  this  group  do  not 
give  an  alcoholic  fermentation  but  do  produce  ether. 


1,  Ascs;  2,  Ascospores;    3,  Germination 
of  Ascospores  according  to  Klocker. 


1  Klocker,  A.  Recherches  sur  les  organismens  de  la  fermentation.  II.  Re- 
cherches  sur  17  formes  des  Saccharomyces  apiculatus.  Comp.  Rend.  Trav.  lab. 
Carlsberg,  1913. 


276  FAMILY  OF  SACCHAROMYCETACEAE 

Genus  XI 

Spherical  or  hemispherical  ascospores,  irregular  or  angulous  in 
form.  Generally  no  fermentation  is  produced.  Forms  a  strong  my- 
celium. 

PICHIA  MEMBRANAEFACIENS.    Hansen 
Syn.:  SACCHAROMYCES  MEMBRANAEFACIENS.     Hansen 

This  species  was  found  by  Hansen  in  the  gummy  exudations  of 
the  elm,  by  Koehler  in  well  water  and  by  Jorgensen  in  white  wine. 
It  has  been  described  by  Hansen  l  and  Siefert.2  It  resembles  the 
Mycodermae  (Mycoderma  cerevisiae,  or  vini).  When  cultivated  on 
beer  wort  a  thick  scum  is  rapidly  formed,  folded  and  grayish  in  color. 
It  is  filled  with  air  bubbles.  The  cells  are  spherical  or  elongated  and 
rich  in  vacuoles  (Fig.  130).  The  temperature  limits  for  budding  in 
beer  wort  are:  minimum,  0.5°  C.  and  maximum,  35°  C.-360  C.  The 
scum  is  not  formed  at  the  temperature  limits  and  the  yeast  vegetates, 
then,  as  a  deposit. 

The  ascs  form  easily  on  plaster  blocks  and  also  in  most  of  the 
culture  media  and  especially  in  the  scums.  They  possess  two  spheri- 
cal, elongated,  hemispherical  or  oval  ascospores,  sometimes  trian- 
gular, which  measure  about  4.5  microns  in  length.  According  to 
Nielsen,3  the  maximum  temperature  for  budding  is  situated  between 
33  and  35°  C.,  the  minimum  temperature  between  2.5  and  2.7°  C.  and 
the  optimum  between  30.5  and  31°  C.  At  this  latter  temperature  the 
ascospores  are  formed  in  19-21  hours.  On  wort  gelatin,  the  colonies 
develop  on  the  surface  of  the  substratum  and  look  like  a  shield. 
They  are  rugose  and  have  a  reddish  tint.  The  gelatin  is  liquefied 
very  rapidly.  The  colonies  which  develop  in  the  depths  of  the  me- 
dium have  a  different  appearance  and  liquefy  gelatin  less  rapidly. 
This  species  does  not  invert  saccharose,  and  according  to  Hansen, 
does  not  ferment  any  sugar.  However,  Lindner  has  caused  a  slight 
fermentation  of  dextrose  and  levulose. 

1  Hansen,  E.  C.     Recherches  sur  la  physiologie  et  la  morphologic  des  fer- 
ments alcooliques.     VII,  Action  des  ferments  alcooliques  sur  les  diverses  especes 
de  sucre.    Levures  alcooliques  a  cellules  ressemblant  a  des  Saccharomyces.    Comp. 
Rend.  du.  lab.  de  Carlsberg,  2,  Book  5,  1888;    XI,     La  spore  de  Saccharomyces 
devenue    sporage.      Recherches    comparatives    sur    les  conditions    de    croissance 
vegetative  et  le  developpement  des  organes  de    reproduction   des  levures  et   des 
moisissures.    Comp.  Rend,  du  Lab.  de  Carlsberg,  5,  Book  2,  1902. 

2  Siefert,  W.     Saccharomyces  membranaefaciens.     Ber.  d.  chem.  Phys.  Ver- 
suchsst.    Jlosternburg,  1899-1900. 

3  Nielsen,  J.  C.  Sur  le  developpement  des  spores  du  Saccharomyces  membranae- 
faciens, du  Sacch.  Ludwigii,  et  du  Sacch.  anomalous.     Comp.  Rend,  du  lab.  de 
Carlsberg,  3,  Book,  31,  1891. 


PICHIA  HYALOSPORA 


277 


PICHIA  MEMBRANAEFACIENS  II  AND  III.    Pichi 
Syn.:  SACCHAROMYCES  MEMBRANAEFACIENS  n  AND  in.     Pichi 

Pichi 1  has  described  two  species  of  yeast  which  very  much  re- 
semble P.  membranaefaciens  and  which  he  has  called  Saccharomyces 
membranaefaciens  II  and  Saccharomyces  membranaefaciens  III.  These 
two  species  have  somewhat  the  same 
physiological  and  morphological  charac- 
teristics of  Saccharomyces  membranae- 
faciens  of  Hansen.  They  seem  to 
differ  only  in  the  shape  of  their  ascs. 
P.  membranaefaciens  II  was  found 
on  the  leaves  of  Evonymus  europaeus. 
The  cells  are  usually  5  to  7  JJL  long  and 
3.5  jit  wide  and  even  10-19  jit  long  and 
3-4  to  5  fJL  wide.  The  ascospores  are 
round  or  a  little  flattened  (2.5-3  ju). 
The  ascs  enclose  3-4  ascospores  and 
form  in  a  milky  white,  folded  scum. 
They  are  oval  (6-8  JU  long  by  3-5  fJL  Fig.  130.  —  Pichia  membranaefaciens 
wide). 

P.  membranaefaciens  III  was  isolated  from  wine.     The  cells  are 
5  to  7  ju  long  and  3-4  to  6  ^  wide.    The  ascospores  (2.5  X  3.5  p)  are  to 

the  number  of  2-4  in  a  spherical  or 
oval  asc.  The  scum  develops  on  beer 
wort  at  22-25°  C.  and  is  folded  with 
a  large  number  of  ascs. 


0  ®;@;@', 


PICHIA  HYALOSPORA.    Lindner 

Syn.:  SACCHAROMYCES  HYALOSPORUS. 
Lindner 

This  yeast,  discovered  by  Lindner, 
forms  on  beer  wort  a  delicate  scum. 
The  ascospores  are  rounded,  each  being 
provided  with  a  brilliant  granule  in 
p  ,  the  center  probably  of  the  nature  of 
on  Saccharose"  Gelatin.  Vegeta-  a  fat  globule  (Fig.  131).  The  giant 
live  Cells  and  Ascs  (after  Lindner).  colonies  are  a  dull  gray  with  a  filaceOUS 

appearance.     This  yeast  does  not  induce  a  fermentation. 

1  Pichi,  P.  Ricierche  morph.  e  fisilogische  sopra  due  nuove  specie  di  Sacch. 
prossime  al  S.  membranaefacfens.  Ann.  della  R.  Scuola  di  viticoltura  e  di  Enologia 
in  Conegliano,  1,  1892. 


278  FAMILY  OF  SACCHAROMYCETACEAE 

PICHIA  TAURICA.     Siefert 
Syn.  SACCHAROMYCES  MEMBRANAEFACIENS,  var.  TAURicus.    Siefert 

This  species  was  found  by  Siefert l  in  Crimean  wine.  It  forms  a 
delicate  scum  which  falls  to  the  bottom  of  the  culture  flask  very 
easily.  This  scum  is  composed  of  elongated  or  sausage-shaped  cells 
(20  IJL  long  and  4-6  JJL  wide)  rarely  oval.  The  temperature  limits  for 
budding  are:  maximum,  22°  C.,  minimum,  5-6°  C.  on  wine  with  8  per 
cent  of  alcohol.  The  optimum  is  about  22°  C.  Numerous  ascs  are 
formed  after  a  short  time  in  the  scum.  The  temperature  limits  for 
sporulation  are  5-6°  C.  and  34°  C.  This  yeast  does  not  grow  in  more 
than  12.2  per  cent  of  alcohol  by  volume. 


PICHIA  TAMARINDORUM.    Siefert2 
Syn:  SACCHAROMYCES  MEMBRANAEFACIENS  var.  TAMARINDORUM.  Siefert 

Isolated  from  an  alcoholic  drink  prepared  with  tamarind,  this 
yeast  possesses  elongated  cells  rarely  oval  or  pear  shaped,  with  a  very 
refractive  granule  in  the  protoplasm.  The  elongated  cells  are  about 
2-6  p,  in  length  and  2-6  ju,  in  width.  The  oval  cells  are  about  5-6  fj,  in 
length  and  2-3  JJL  in  width. 

The  scum  is  thick  powdery  white,  and  wrinkled  in  old  cultures. 
If  it  is  disturbed,  it  falls  to  the  bottom  of  the  flask  in  large  flakes. 
Spores  are  abundantly  produced  in  the  scum  at  temperatures  of  the 
laboratory.  They  are  also  produced  easily  on  plaster  blocks  between 
27  and  30°  C.  At  34°  C.  and  at  1.5°  C.  no  ascospores  are  formed.  The 
ascospores  are  hemispherical  (3  to  4  ju)  and  usually  possess  a  refractive 
granule  in  the  center.  On  gelatin  the  giant  colonies  have  a  charac- 
teristic rosette  appearance. 


PICHIA  CALIFORNICA.    Siefert 
Syn.:  SACCHAROMYCES  MEMBRANAEFACIENS  var.  CALIFORNICUS.    Siefert1 

Discovered  by  Siefert  in  red  wine  from  California,  this  yeast 
generally  possesses  oval  cells  (4-8  ju  in  length  and  3-5  JJL  in  width) 
enclosing  a  refractive  body.  It  forms  a  delicate  scum  which  falls  to 

1  Siefert,    W.      Ueber   die   Sauerabnahme   im  Wein   und  den   dabei   stattfin- 
denden  Garungsprozess.     Zeit.  landw.  Versuchswesen  in  Osterreich.     1900. 

2  Siefert,    W.      Saccharomyces    membranaefaciens.      Ber.    d.    Chem.    physiol. 
Versuchsst.  Klosterneuburg.    6,  1899-1900. 


PICHIA  FARINOSA  279 

the  bottom  of  the  flask  when  disturbed.  The  temperature  limits 
for  budding  are  7-12°  C.  and  33°  C.  in  wine  with  8  per  cent  of  alcohol. 
The  optimum  is  situated  between  28°  and  30°  C.  In  beer  wort  the 
maximum  temperature  is  near  39°  C.  The  temperature  limits  for 
sporulation  are  5-6°  C.  and  39-40°  C.  The  optimum  is  near  34°  C.  The 
ascospores  .are  spherical  and  refractive  (2-3  p  in  diameter)  and  de- 
velop only  in  12.2  per  cent  of  alcohol  by  volume.  This  species  forms 
less  glycerol  in  Pasteur's  medium  and  does  not  attack  alcohol  as 
much  as  P.  membranaefatiens.  Pichia  californica  has  also  been  found 
by  Saito  l  in  the  fermentation  of  rum  from  molasses  on  the  island  of 
Bonin,  Japan. 

PICHIA  RADAISH.     Lutz 
Syn.:  SACCHAROMYCES  RADAISH.    Lutz  2 

This  species  was  isolated  by  Lutz  from  tivy,  an  alcoholic  drink, 
prepared  in  Mexico.  It  has  oval  or  elongated  cells  (8  to  8.5  IJL  long 
and  3  to  3.5  p  wide).  The  optimum  temperature  for  the  formation 
of  a  scum  is  23°  C.  Budding  ceases  at  about  37-38°  C.  Sporulation  is 
accomplished  at  the  end  of  12  hours  at  22°-23°  C.  The  maximum  tem- 
perature for  the  formation  of  ascospores  is  between  25  and  28°  C. 
The  ascospores  (1.4/x  in  diameter)  are  spherical  and  generally  to  the 
number  of  4  per  asc.  This  species  does  not  liquefy  gelatin.  The  col- 
onies are  reddish  in  color  which  appear  after  a  culture  period  of  some 
time. 

PICHIA  FARINOSA.     Lindner 
Syn.:  SACCHAROMYCES  FARINOSUS.     Lindner 

This  species  was  found  by  Lindner 3  in  beer  in  Danzig.  Saito  has 
recently  found  it  among  the  yeasts  in  soy  bean  sauce.  The  cells  are 
generally  elongated;  some  are  ellipsoidal  (Fig.  127).  The  maximum 
temperature  for  the  formation  of  the  scum  is  37°  C.  On  beer  wort,  or 
decoction  of  "  koji,"  it  forms  a  dry  scum  after  24  hours.  Ascospores 
are  formed  easily  in  most  media  notably  in  the  scum,  but  the  prop- 
erty of  forming  spores  is  completely  lost  by  cultivating  on  gelatin. 
The  ascospores  are  to  the  number  of  1  to  4  per  asc.  They  are  rounded 
or  oval  with  a  brilliant  granule  in  the  center.  According  to  the  il- 
lustrations of  Lindner  and  Saito  it  seems  that  the  ascs  are  derived 

1  Saito,  K.     Notiz  iiber  die  Melasse-rumgarung  auf  den  Bonin-inseln.  (Japan). 
Cent.  Bakt.  16,  1908. 

2  Lutz,  C.     Rech.  sur  la  constitution  du  Tiby.     Bull.  Sic.  myc.  de  France, 
15,  1899. 

3  Lindner,   P.     Saccharomyces  farinosus   and   Bailii.     Wochensch.   f.    Brau. 
1893. 


280 


FAMILY  OF  SACCHAROMYCETACEAE 


from  a  copulation.  These  authors  show  ascs  formed  from  two  cells 
united  by  a  canal.  Sometimes  only  one  will  produce  ascospores  and 
sometimes  both.  (Fig.  132.)  Guilliermond  l  has  shown  that  these 
illustrations  simply  represent  budding.  In  fact,  the  cells  destined  to 
sporulate  are  able  to  continue  budding  at  the  moment  of  sporula- 
tion  and  the  ascospores  often  form  before  the  separation  of  the  cell 

which  is  formed  in  the  bud- 
ding process.  Sometimes 
the  bud  will  form  on  the 
lateral  surface  of  the  cell, 
then  will  elongate  parallel 
with  the  mother  cell  which 
results  in  forms  resembling 
the  Zygosaccharomyces. 

The  giant  colonies  on 
maltose  gelatin  are  circular 
and  have  a  chalky  white 
appearance .  On  g  e  1  at  i  n 
streaks,  the  culture  is  also 

Fig.   132.-Pichia  farinosa.     Vegetative  Cells    whitish  and   the  edSe  finely 
and  Ascs  in  Scum  on  Beer  Wort  (after  Lind-    indented.       Liquefaction    of 

ner'*  the  gelatin  is  quite    rapidly 

accomplished.  The  giant  colonies  are  chalky  white  with  a  folded 
surface  and  slowly  liquefy  the  gelatin.  This  yeast  feebly  ferments  dex- 
trose and  levulose  but  has  no  action  on  other  sugars  (maltose,  d-galac- 
tose,  saccharose,  melibiose,  raffinose,  a-methylglucosides).  In  beer 
wort,  the  amount  of  alcohol  produced  is  very  small.  In  11  days,  0.9 
per  cent  of  alcohol  and  ethyl  ether  is  formed.  According  to  Siefert, 
Pichia  farinosa  produces  in  alcoholic  must  acetic  acid. 


PICHIA  SUAVEBLEUS.     Klocker 

Found  in  soil  in  Denmark,  this  species  forms 
on  must  at  the  end  of  24  hours  at  25°  C.  a  gray 
scum  which  adheres  to  the  walls  of  the  flask,  and 
a  white  thick  ring.  On  beer  wort  the  scum  is 
thick  and  compact  with  a  yellowish  color.  The 
cells  in  the  scum  are  spherical  or  oval  (5  to  8ju 
long).  The  temperature  limits  for  the  formation 
of  scum  are  34-36°  C.  and  10-4°  C.  The  ascospores 
are  spherical  sometimes  flattened  on  one  side  with 

1  Guilliermond,  A.    Quelques  remarques  sur  la  sexualite  des  levures.    Annals, 
mycologici,  8,  1910. 


Fig.  132-bis.  —  Pichia 
suavebleus.  Cells  from 
a  Young  Scum  on 
Beer  Wort  (after 
Klocker). 


PICHIA  CALLIPHORA 


281 


a  large  refractive  globule  in  the  center.  Each  asc  contains  about  2 
ascospores.  Ascospores  are  formed  with  difficulty  on  plates.  The 
temperature  limits  for  sporulation  on  plates  are  29-33°  C.  and  10°  C. 
It  ferments  dextrose  and  saccharose  only.  In  beer  wort  it  sets  up  a 
feeble  fermentation  with  a  fruity  odor.  Gelatin  is  not  liquefied. 


PICHIA  ALCOHOLOPHILA.     Klocker 

This  species  was  found  in  Denmark.  On  must  after  a  few  days  it 
forms  a  scum  at  25°  C.  more  or  less  developed.  On  must  with  10  per 
cent  of  alcohol  added,  it  forms  a  well-developed 
scum.  It  is  folded  and  adheres  to  the  walls 
of  the  flask.  There  is  abundant  sediment  al 
growth.  The  cells  in  the  scum  are  spherical; 
those  of  the  sediment  are  oval  or  sausage 
shaped.  The  temperature  limits  for  the  forma- 
tion of  the  scum  are  33-35°  C.  and  3.4  to  8.4° 

C.     The  ascospores  are  to  the  number  of  four, 

m,  .     J 

sometimes  two  per  asc.     They  are  spherical 

or   hemispherical.     They    are    formed    abun- 

,,     .  rnu     v     -x       r  x 

dantly  in  the  scums.     The  limits  of  tempera- 

ture for  the  formation  of  spores  on  plates  are  29-33°  C.  and  0.5-4°  C. 
Gelatin  is  partially  liquefied.     Dextrose  is  feebly  feimented. 


if-  713?77A-  ~  Pic^a 
holophila  (after  Klocker). 

Cells  from  a  Young  Scum 
on  Beer  Wort  to  which 
Alcohol  was  Added. 


PICHIA  POLYMORPHA.     Klocker 

This    species   was    also    found    in    the    soil    of 
Denmark.     A  white  well-developed  scum  is  formed 
on  must  in  24  hours  at  25°  C.     It  adheres  to  the 
sides  of  the  flask.    At  first  the  cells  are  elongated 
or  egg-shaped    (13   JJL  long).     They  bud   laterally. 
Fig.  132-B.  —  Pichia  Finally,  they  become  spherical  or  oval.    The  tem- 
polymorpha    (after  perature  limits  for  scum  formation  are  39°  and  0.5° 
from  it   Young  C-     The  ascospores  are  spherir 
Scumon  Beer  Wort   cal  and  formed  with  difficulty 
on  plates.     Gelatin  is  liquefied. 
Dextrose  and  saccharose  are  fermented  and  maltose 
feebly. 


Fig.  132-C.  —  Pichia 
calliphora  (after 
Klocker).  Cells  from 
a  Young  Scum  on 
Beer  Wort  at  25°  C. 


PICHIA  CALLIPHORA.     Klocker 

This  species  was  found  on  the  fly  Calliphora 
arthrocephala  at  Carlsberg.    On  beer  wort,  a  white 
scum  is  formed  along  with  a  feeble  ring.     The  cells  are  sausage  shaped 
and  almost  13  /it  long.     The  temperature  limits  for  scum  formation  are 


282 


FAMILY   OF  SACCHAROMYCETACEAE 


33-35°  C.  and  0.5-4°  C.  The  ascospores  are  small,  spherical  or  hemi- 
spherical to  the  number  of  2  to  4  per  asc.  The  temperature  limits 
of  sporulation  on  plates  are  24-27°  and  7-10°  C.  Gelatin  is  liquefied. 
A  fermentation  is  set  up  in  wort.  Dextrose  only  is  fermented. 

PICHIA  MANDSHURICUS.    Saito 

This  was  isolated  from  Chinese  yeast  used  in  the  preparation  of 
an  alcoholic  drink  in  Manchuria.  It  was  found  along  with  Zygosac- 
charomyces  mandshuricus.  The  temperature  limits  for  scum  formation 

are  7-10°  C.  and  40-41°  C.  The 
spores  are  globular  or  spherical 
(2-4  /A)  obtained  in  the  scums  on 
plaster  blocks,  1  to  4  per  asc.  The 
temperature  limits  for  sporulation 
are  11-16-32°  C.  Dextrose  is  fer- 
mented. 

At  the  same  time  Saito  isolated 
another  species  of  the  same  group 
Fig.  132-D.  —  Pichm^marrtshuricus  (after   named    Kocoling    chiu    Kahmhefe 

which  has  elongated  cells.      The 

temperature  limits  for  the  formation  of  the  scum  are  7-10°  C.  and 
60°  C.  It  ferments  dextrose,  fructose  and  mannose.  Only  once  on 
Gorodkowa's  gelatin  did  the  author  observe  spore  formation. 

YEAST  FROM  PULQUE  NO.  1.  Guilliermond  * 

This  species  was  isolated  from  the  fermentation  of  Pulque.  It 
belongs  to  the  genus  Pichia.  On  beer  wort,  at  25-30°  C.,  there  are 
formed  at  the  end  of  a  day,  small  floating  patches  on  the  surface  of 
the  liquid  and  after  48  hours,  a  scum  which 
is  grayish  white  in  color.  A  sedimental 
growth  is  also  formed.  The  cells  are  gener- 
ally oval,  sometimes  round.  In  old  cultures, 
they  are  elongated  and  remain  in  chain 
formations.  The  maximum  temperature  for 
growth  is  near  30°  C.,  the  optimum  being 
between  29°  C.  and  30°  C.  The  ascospores 
appear  on  most  media.  They  are  to  the 
number  .of  1  to  4  per  asc,  and  have  a  hemispherical  shape.  The  maxi- 
mum temperature  for  sporulation  is  around  38-39°  C.  The  optimum 
is  between  29-32°  C.  The  yeast  inverts  saccharose  but  produces  no 
fermentation. 

1  Guilliermond,  A.  Levaduras  del  Pulque.  Boletin  de  la  Direction  de  Etudios 
Biologicos,  Mexico,  2,  1917. 


i^-E,  —Yeast    from 
Pulque  No.  1.    Cells  from 


Carrot 


(after     Guillier- 


SACCHAROMYCES   MYCODERMA  PUNCTISPORUS    283 


•      PICHIA  ORIENTALIA.    Beijerinck l 

This  yeast  is  little  known  but  seems  to  be  related  to  the  genus 
Pichia.  It  possesses  ascs  which  makes  it  improper  to  class  it  with 
the  Pichia.  It  was  isolated  by  Beijerinck  from  a  sample  of  "  koji " 
secured  from  Eyckman.  The  same  author  has  found  it  on  certain 
oriental  fruits  (grapes  from  Corinth).  It  is  a  yeast  which  especially 
ferments  dextrose.  In  yeast  water  to  which  glucose  has  been  added, 
it  brings  about  an  active  fermentation.  In  beer  wort  the  fermenta- 
tion is  less  active.  It  does  not  ferment  maltose.  On  beer  wort  it 
forms  a  scum  and  produces  carbon  dioxide.  In  culturing  it  on  grape 
must  to  which  lactic  acid  has  been  added,  at  28°  C.,  it  produces  a  dry 
scum,  powdery,  in  which  numerous  ascospores  are  formed. 


SACCHAROMYCES  MYCODERMA  PUNCTISPORUS.    Melard2 

This  yeast  which  seems 'to  belong  to  the  Pichia  was  isolated  by 
Melard  in  a  Belgian  brewery  from  a  top  fermentation.  It  causes  a 
disagreeable  taste  in  beer.  The  cells  belong  to  the  Pastorianus  or 
ellipsoideus  type  and  show,  in  their  interior,  from  1  to  3  small  black 
points.  The  yeast  is  essentially  aerobic  and  develops  a  scum  rapidly 
on  liquid  media.  This  scum  varies  with  the  medium.  It  may  pre- 
sent a  scum  like  ordinary  yeasts  or  be  folded  and  present  character- 
istics of  the  scum  of  Mycoderma.  Sporulation  has  been  obtained  on 
plaster  blocks  and  in  scums.  At  the  temperature  of  15-18°  C.  it  is 
accomplished  in  5  to  6  days.  The  ascospores  may  result  from  an  in- 
crease in  size  of  the  black  spots  in  each  cell.  The  giant  colony  is 
dull  and  of  a  yellowish  tint.  It  does  not  liquefy  gelatin.  This  species 
does  not  produce  any  liquefaction.  Levulose  is  preferred  over  other 
sugars  as  a  food.  It  does  not  invert  saccharose. 


Genus  XII.     Willia.  Hansen 

Ascospores  in  the  shape  of  a  lemon  or  hat  with  a  projecting  ring 
around  them.  Most  of  the  species  produce  ethers  but  some  give 
only  an  alcoholic  fermentation. 

1  Beijerinck,   W.     Ueber  regeneration    der  Sporenbildung    bei   Alkoholhefen 
wo  diese  Funktion  in  Verschwinden  begriffen  ist.     Cent.  Bakt.  4,  1898. 

2  Melard,  L.    Note  sur  un  organisme  isole  d'une  biere  de  fermentation  haute. 
First  International  Congress  of  Brewing,  Brussels,  1910 


284  FAMILY  OF  SACCHAROMYCETACEAE 

WILLIA  ANOMALA.     Hansen 
Syn.:  SACCHAROMYCES  ANOMALOUS.     Hansen 

This  species  was  isolated  by  Hansen  l  from  an  impure  brewery 
yeast  which  came  from  Bavaria.  The  cells  of  this  yeast  resemble 
those  of  Torula  discovered  by  Hansen.  They  are  small  cells,  gen- 
erally oval,  often  sausage  shaped.  In  the  beginning  of  fermentation, 
this  yeast  forms  a  dull  gray  scum  which  resembles  very  much  that  of 
Monilia  Candida.  Among  the  cells  in  the  scum  are  found  many  air 
bubbles.  The  temperature  limits  for  budding  on  beer  wort  are 
0.5-1°  C.  and  37-38°  C.  At  these  limits  of  temperature  no  scum  is 
formed.  The  yeast,  then,  develops  as  a  sediment. 

Ascospores  appear  very  easily  at  the  end  of  a  short  time,  as  well 
in  the  scum  as  in  the  cells  in  the  deposit.  They  are  formed  easily 
in  most  solid  media  in  most  favorable  conditions  of  nutri- 
tion. The  temperature  limits  for  sporulation  on  plaster 
blocks  are,  according  to  Nielsen,  32-34°  C.  and  2.5-7.5° 
C.  The  optimum  is  at  30°  C.  At  this  temperature  the 
ascospores  begin  to  form  in  17-18  hours. 

The  number  of  ascospores  varies  from  2  to  4  in  each 
asc.  They  may  be  located  in  the  asc  in  a  diversified 
manner.  Their  diameter  is  about  2  to  3/z.  They  pos- 
sess a  characteristic  shape  absolutely  analogous  to  those 
of  Endomyces  decipiens,  Endomyces  fibuliger  and  Ascoidea 
Fig.  133.  —  rubescens.  They  are  hemispherical  and  shaped  like  a 

faCSammala  hat-      (FiS-  133-)     The  wal1  of  the  asc  is  broken  easily. 

(after  Han-  Germination  of  the  ascospores  is  brought  about  in  the 
following  manner.  The  ascospore  swells  up  during  which 
the  projecting  collar  may  persist  or  disappear  completely.  The  asco- 
spore produces,  at  different  points  on  its  surface,  buds  which  multiply 
in  their  turn  by  budding.  (Fig.  38.)  This  yeast  ferments  beer  wort 
rapidly.  During  the  fermentation  the  liquid  is  stirred  up  becoming 
opalescent,  and  gives  off  a  fruity  odor.  This  species  ferments  dex- 
trose but  not  saccharose  or  maltose.  Willia  anomala  has  been  found 
by  Klocker  and  Schionning,  Kozai  and  Saito  in  "koji  "  employed  in 
the  preparation  of  awamori,  an  alcoholic  drink  on  the  island  of  Luchu. 

BIOLOGICAL  VARIETIES  OF  WILLIA  ANOMALA 

Since  the  discovery  of  this  species,  numerous  varieties  of  the  type 
anomalous  have  been  observed  all  of  which  have  special  characteris- 

1  Hansen,  E.  C.  Recherches  sur  la  physiologic  et  morphologic  des  alcooliques 
ferments.  VIII.  Sur  la  germination  des  spores  chez  les  saccharomyces.  Comp. 
Rend,  du  lab.  de  Carlsberg  3,  1891;  5,  1902. 


WILLIA  ANOMALA  I  285 

tics,  such  as  the  special  shape  of  their  cells  or  the  odor  which  they 
give  off  in  sugar  solutions.  Zeidler  has  described  it  in  the  juice  of  the 
marsh  mallow  and  Jorgensen  in  an  English  yeast.  Beijerinck  has 
described  under  the  name  of  Saccharomyces  acetaethylicus  a  species 
producing  ethyl  ether  which  seems  to  be  a  member  of  the  Willia 
anomala  group.  The  same  author  isolated  a  variety  of  Willia  anomala 
which  he  called  Mycoderma  pulverulenta;'  these  two  species  have  been 
insufficiently  described.  Finally,  Fischer 
and  Brebeck  have  encountered  another 
variety  under  the  name  of  Endoblastoderma 
which  forms,  like  the  Willia  anomala  of 
Hansen,  a  white  powdery  scum,  but  differs 
in  the  method  of  formation  of  endogenous  Fig  i^  —  Wima  anomala  of 
germ.  In  certain  cells  it  forms  a  sort  of  Zeidler  on  Wort  Gelatin  (after 
internal  spore  which  is  placed  well  against 

the  wall  of  the  cell.  This  remains  attached  to  the  cell  like  a  bud  after 
it  has  been  released  from  it.  Klocker  has  shown  that  these  endogenous 
formations  are  due  to  an  optical  illusion  caused  by  budding  and  that 
Endoblastoderma  of  Fischer  and  Brebeck  does  not  differ  from  Willia 
anomala  but  may  be  identified  with  it.  Other  authors  (Holm, 
Meissner,  Will,  Lindner)  have  found  forms  related  to  Willia 
anomala.  Beauverie  and  Lesieure  have  isolated  one  from  spu- 
tum in  pulmonary  tuberculosis.  Finally,  Steuber  l  has  described  a 
series  of  biological  varieties  of  W.  anomala  which  we  shall  describe 
briefly. 

WILLIA  ANOMALA  I.    Steuber 

Syn.:  SACCHAROMYCES  ANOMALUS  i.     Steuber 

This  variety  is  characterized  by  the  aromatic  odor  produced  by 
ethyl  ether  in  its  cultures.  On  beer  wort,  it  forms  a  scum  at  first 
folded  and  yellow.  The  temperature  limits  for  the  formation  of  a 
scum  are  5-10°  C.  and  37°-42°  C.  The  optimum  is  30°  C.  Sporulation 
is  accomplished  on  plaster  blocks  and  rarely  in  scums  or  giant  colonies. 
The  temperature  limits  for  sporulation  on  plaster  blocks  are  5—12°  C. 
and  30°-35°  C.  Ascospores  appear  at  the  end  of  13  to  14  hours  at 
the  optimum  temperature  on  plaster  blocks.  They  rarely  form  in 
scums  and  giant  colonies.  The  ascospores  have  the  form  of  a  hat. 
The  giant  colony  is  yellow  in  the  center  and  white  or  shiny  at  the 
edge.  Gelatin  is  liquefied.  This  variety  ferments  10  per  cent  solu- 
tions of  saccharose,  dextrose  and  levulose.  It  does  not  act  on  mal- 
tose, lactose  or  d-galactose  which  it  uses  in  its  nutrition.  In  liquids 

1  Steuber,  L.  Beitrage  zur  Kentniss  des  Gruppe  Saccharomyces  anomalus. 
Zeitsch.  d.  ges.  Brau.  23,  1900. 


286  FAMILY  OF   SACCHAROMYCETACEAE 

containing  sugars  which  it  ferments,  it  forms  ethyl  ether  and  acetic 
acid,  sometimes  a  little  butyric  acid. 


WILLIA  ANOMALA  H.    Steuber 
Syn.  SACCHAROMYCES  ANOMALUS  ii.     Steuber 

This  variety  forms  on  beer  wort  a  scum  which  is  at  first  folded  and 
chalky  but  which  later  assumes  a  rose  tint.  The  temperature  limits  for 
the  formation  of  a  scum  are  5  to  10°  C.  and  30°-35°  C.  The  optimum 
is  about  30°  C.  The  formation  of  ascospores  is  easily  accomplished  on 
media  in  44  hours/  The  temperature  limits  for  sporulation  on  plaster 
blocks  are  5°-15°  and  30°-35°  C.  The  ascospores  are  hat  shaped. 
After  a  certain  time  the  giant  colonies  are  rose  colored  or  reddish 
brown.  It  liquefies  gelatin.  This  variety  inverts  saccharose  which  it 
ferments  slowly  but  completely.  It  produces  only  traces  of  alcohol 
in  10  per  cent  solutions  of  levulose.  It  has  no  action  on  other  sugars. 
It  does  not  form  ether  or  fatty  acid  but  traces  of  acetic  and  butyric 
acids. 

WILLIA  ANOMALA  HI.    Steuber 
Syn.:  SACCHAROMYCES  ANOMALUS  in.     Steuber 

This  variety  came  from  brown  Munich  beer.  The  scum  is  white, 
later  yellow.  The  temperature  limits  for  its  formation  are  5-15°  C. 
and  30-35°  C.  The  optimum  is  30°  C.  The  temperature  limits  of  sporu- 
lation on  plaster  blocks  are  5-15°  C.  and  30-35°  C.  The  ascospores 
form  in  great  numbers  in  giant  colonies  but  never  in  scums.  They 
are  hat  shaped.  The  giant  colony  is  white,  irregular  and  liquefies 
gelatin.  This  variety  gives  only  traces  of  alcohol  in  solutions  of  10 
per  cent  levulose  and  does  not  ferment  any  other  sugar.  It  produces 
neither  ethyl  ether  nor  fatty  acids  but,  at  the  beginning,  only  traces 
of  acetic  acid  and  butyric  acid  which  are  eventually  oxidized. 

WILLIA  ANOMALA  IV.     Steuber 
Syn.:  SACCHAROMYCES  ANOMALUS  iv.     Steuber 

This  variety  has  the  same  origin  as  the  preceding  variety.  The 
scum  is  white  and  later  yellow.  Its  temperature  limits  are  5-15°  C. 
and  35-41°  C.  The  temperature  limits  for  sporulation  are  15-20°  C. 
and  30-35°  C.  The  ascospores  are  hat-shaped.  The  sporogenic  prop- 
erty is  lost  on  long  cultivation  while  it  is  preserved  in  the  preceding 
variety.  This  is  one  method  for  distinguishing  between  them.  The 
giant  colony  is  at  first  white  and  later  yellow  and  folded.  It  liquefies 


WILLIA  ANOMALA  287 

gelatin.  This  variety  forms  traces  of  alcohol  in  solutions  of  10  per 
cent  levulose  and  acts  on  no  other  sugar.  From  the  point  of  view  of 
acids  and  ether  it  acts  like  the  preceding  yeast. 

WILLIA  BELGICA.    Lindner 
Syn.:  SACCHAROMYCES  ANOMALUS    var.  BELGICUS.    Lindner  l 

This  yeast,  closely  related  to  Willia  anomala,  was  discovered  by 
Lindner  in  Belgian  beer.  The  cells  are  small  with  thin  walls.  This 
species  produces  ascospores  which  look  like  those  of  Willia  anomala. 
It  is  distinguished  from  Willia  anomala  by  the  fact  that  it  ferments 
not  only  dextrose  but  d-mannose,  d-galactose  and  levulose  and  that 
it  does  not  produce  an  ether  odor.  It  develops  on  beer  wort  in  the 
form  of  a  dotted  scum. 

WILLIA  WICHMANNI.     Zikes2 

Isolated  from  soil  near  Vienna  this  yeast  has  cells  3  to  5/4  rang- 
ing from  6  to  40  ju,  long  in  scums.  It  grows  on  agar  from  5°  C.  to  42°  C. 
The  optimum  is  22°  C.  Sporulation  appears  rapidly  at  21°  C.  on  plaster 
blocks,  less  actively  at  18  to  28°  C.  The  ascospores  are  hat  shaped 
(2ju  in  diameter)  and  are  to  the  number  of  2,  3  or  4  per  asc.  This 
yeast  grows  on  peptone  gelatin  in  a  white  layer.  On  glucose  agar, 
the  colonies  look  like  little  droplets,  whitish  yellow  in  color.  On 
potato,  the  yeast  gives  a  grayish  viscous  growth.  On  slices  of  beet, 
it  produces  a  yellow  layer  and  abundant  development  with  the  for- 
mation of  numerous  ascospores.  It  causes  a  cloudy  beer  wort  with 
the  formation  of  a  scum  in  3  days.  This  yeast  ferments  dextrose  and 
levulose  but  does  not  attack  maltose,  d-mannose,  d-galactose,  lactose, 
saccharose,  raffinose,  inuline  or  dextrine.  It  forms  ethyl  ether  in 
fermentation. 

WILLIA  ANOMALA.    Saito 

This  yeast  forms  a  thick  white  scum  on  must,  white  and  adher- 
ing a  little  to  the  walls  of  the  container.  The  cells 
are  round  or  oval  and  sometimes  in  chain  formation. 
The  giant  colonies  on  gelatin  are  white  and  united. 
The  temperature  limits  for  scum  formation  are: 
maximum,  35°  C.  and  minimum,  2-3°  C.  The  spores 
are  shaped  like  a  hat  with  a  projecting  edge  (2.7-  Fig.  134-A.— Ascsof 
3.6 ju  in  diameter).  They  are  to  the  number  of  4  Willia  anomala, 
per  asc.  They  form  abundantly  on  plaster  blocks 

1  Lindner,    P.      Mikroskopische    Betriebskontrolle    in    den    Garungswerben, 
Paul  Parey,  Berlin,  6th  edition,  1909. 

2  Zikes,  H.    Ueber  anomalus-Hefe  und  eine  neue  Art  derselben.     Cent.  Bakt. 
16,  1906. 


288  FAMILY  OF  SACCHAROMYCETACEAE 

but  in  scums  on  beer  wort  they  appear  very  rarely.  The  temperature 
limits  for  sporulation  are  2-3°  C.  and  30°  C.  This  yeast  ferments 
dextrose,  levulose,  saccharose,  mannose  and  raffinose. 

WILLIA  SATURNUS.     Klocker 
Syn.:    SACCHAROMYCES  SATURNUS.     Klocker 

This  yeast  was  found  by   Klocker  l  in  a  sample  of  earth  from 

Himalaya.     It  has  since  been  found  in  Italy  and  Denmark.     It  de- 

^  velops  on  must  with  a  white  wrinkled  growth  and  a 

Q,Hu  0 -••  sediment.     In  a  young  scum,  the  cells  are  globular 

"$'Q?      or  oval  in  shape  (4  to  6/x)   (Fig.  135).     Later  they 


enlarge,  become  spherical  and  filled  with  globules  of 
fat.     At  the  same  time  the  scum  increases,   great 
&  bubbles  of  carbon  dioxide  are  formed  and  the  color 

Fig.  135.  —  Willia   becomes  yellow.     If  the  culture  is  shaken  the  scum 

Satwmua.  Cells  of   will  fall  to  the  bottom  of  the  culture  flask  and  a  ring 

Scum     on     Beer    . 

Wort    after    24   is  formed  around  the  culture  flask. 


Hours  at  25°  C.         ^he  temperature  limits  for  budding  on  beer  wort 

are  2  to  4°  C.  and  35°  to  37°  C.     The  optimum  is 

situated   between  28  and  30°  C.     The  scum  forms  easily  on  yeast 

water    to    which   pure   saccharose   is  added.      Maltose,    dextrose    or 

levulose  also  serve  well. 

Sporulation  is  rapidly  accomplished  on  plaster  blocks.     The  tem- 
perature limits  are  4  to  7°  C.  and  28°  to  31.5°  C.    The  optimum  is  near 
25°  C.    At  this  last  temperature,  the  ascospores  appear  at  the  end  of 
43  hours.    They  also  develop  abundantly  in  the  scum 
on  yeast  water  and  rice.     They  appear  both  in  the  ^§y 

ring  in  the  side  of  the  culture  tube  and  in  the  scums  *-*         ^ 

of  cultures  on  must  and  also  in  old  cultures  on  gelatin.          ™"      *3r 
The  shape  of  the  ascospore  is  that  of  a  lemon  but   Fig.   136.  —  Ascs 
sometimes  the  points  are  less  evident  and  indistinct.       ?aftpPVifiS!r^ 

\dtL  Lt?i    xYlUClvtM^  * 

The  ascospore  is  always  girdled  with  a  projecting  ring, 
which  reminds  one  of  the  planet  Saturn,  indicating  the  origin  of  the 
name  of  this  yeast.  In  the  interior  of  the  cells,  there  is  a  globule 
probably  of  fat.  The  length  of  the  ascospore  is  about  3/i  and  the 
width  2//.  The  ascs  include  generally  two  ascospores,  sometimes  three, 
rarely  one,  and  exceptionally  four.  The  germination  of  the  ascospores 
operates  by  budding.  Often  it  is  preceded  by  a  fusion  of  ascospores 
two  by  two  which  is  accompanied  by  a  nuclear  fusion  constituting  a 
true  copulation  (parthenogamy)  (Fig.  39). 

1  Klocker,    A.      Une   espece   nouvelle   de   la    famille  Sacchar.    (S.    saturnus). 
Comp.  Rend,  des  trav.  lab.  de  Carlsberg,  6,  1903. 


WILLIA  SATURNUS  289 

On  gelatin  must,  Willia  saturnus  forms  white  or  pale  yellow 
colonies,  the  surfaces  of  which  are  wrinkled  or  folded.  Their  form  re- 
sembles a  crater.  The  cells  in  the  colonies  have  a  spherical  form  con- 
taining numerous  drops  of  fat.  Gelatin  is  liquefied  slowly.  On  a 
mixed  gelatin  and  peptone  bouillon,  the  cells  are  small  and  often  a 
bit  elongated.  The  appearance  of  the  colony  is  much  like  that  on 
gelatin  must. 

Willia  saturnus  inverts  saccharose.  It  ferments  dextrose,  raf- 
finose,  levulose,  but  has  no  action  on  maltose,  lactose  or  arabinose. 
In  beer  wort,  it  provokes  a  slow  fermentation.  It  gives  off  an  odor 
of  ethyl  ether.  Alcohol  disappears  after  a  time  in  cultures;  probably 
being  oxidized. 

Sartory  has  found  in  the  juice  on  banana  leaves  a  yeast  which  is 
much  like  Willia  saturnus  and  which  may  be  a  variety  of  it.  In 
beer  wort,  the  cells  in  the  sediment  are  oval  (7-8  X  4.5  ju).  The 
optimum  temperature  for  budding  on  carrot  is  situated  between  32 
and  34°  C.;  the  maximum  is  between  41°  C.  and  42°  C.  The  yeast 
forms  a  scum  after  36  hours  in  glycerol  bouillon  at  15-18°  C.,  after 
2  days  at  20-22°  C.,  and  after  3  days  at  38-39°  C.  None  is  formed 
higher  than  this.  The  scum  consists  of  cells  like  those  in  the  sediment, 
but  when  they  become  old  they  are  spring  shaped  or  like  a  pseudo- 
mycelium.  On  beer  wort  gelatin,  the  yeast  forms  a  dull,  white  round 
colony  with  a  banana-like  odor. 

Sporulation  is  accomplished  on  plaster  block  only  on  condition 
that  the  yeast  be  associated  with  the  bacteria  with  which  it  is  found 
and  from  which  it  is  separated  with  great  difficulty.  The  tempera- 
ture limits  for  sporulation  are  a  little  below  8°  and  from  22-30°  C. 
The  optimum  is  situated  between  15  and  18°  C.  The  ascs  (8-9  JJL  in 
diameter)  have  one  to  four  ascospores  (2-3  /z)  similar  in  shape  to  those 
of  Willia  saturnus,  which  germinate  by  ordinary  budding.  The  yeast 
secretes  invertase  and  produces  alcoholic  fermentation. 

Fifth  Group 

Budding  yeasts  with  uncertain  affinities.  Fusiform  ascospores  in 
the  form  of  a  needle. 

Genus  XIII.     Monospora.     Metschnikoff 1 

Single  ascospore  in  the  form  of  a  needle,  germinating  laterally  in 
a  digitiform  prolongation  which  buds  into  dissociated  oval  bodies. 
This  genus  is  represented  by  only  Monospora  cuspidata. 

1  Metschnikoff,  E.  Ueber  eine  Sprosspilzarankheit  der  Daphnien.  Beitrag 
zur  Lehre  uber  den  Krampf  der  Phagocyten  gegen  Krankheitserreger.  Virchow's 
Archives,  96,  1884. 


290  FAMILY  OF  SACCHAROMYCETACEAE 

MONOSPORA  CUSPIDATA.    Metschnikoff 

This  yeast  was  found  by   Metschnikoff  in   1884,  in  the  general 

cavity  of  the  Daphnia.  It  possesses  cells 
which  are  oval  and  which  elongate  to 
form  the  asc.  Each  asc  includes  a  single 
ascospore,  very  thin  and  elongated  in  the 
form  of  a  needle.  It  germinates  by  bud- 
ding on  a  side  with  the  formation  of  oval 
cells.  (Fig.  137.)  The  ascospores  swal- 
lowed by  the  Daphnia  reach  the  intestine 
and  finally  the  general  cavity.  Here  they 
bud  quickly  and  cause  the  death  of  the 
animal.  Metschnikoff,  on  account  of 
Fig.  137.  —  Morwspora  cuspidate,  the  transparency  of  this  organism,  has 
1-7,  Vegetative  Ceils  as  a  Means  of  been  able  to  follow  all  the  steps  in  the 

Budding.     8-10,  Formation  of  the  Asc. 

11,  Germination  of  the  ascospore  (after    prOCCSS  of  phagOCVtOSlS  taking   place  With 

Metschnikoff).  f  ^  .   e      J 

the  cells  derived  from  the  ascospores. 

Genus  XIV.     Nematospora.     Peglion l 

Ascospores  spindle-shaped  with  a  long  cilium  at  one  of  the  ex- 
tremities. Germination  is  accomplished  by  budding  at  both  ends. 
Many  ascospores  in  each  asc.  Up 
to  the  present  only  two  species  are 
known. 


NEMATOSPORA  CORYLI.   Peglion 

This  very  curious  yeast  was  dis- 
covered by  Peglion  in  Italy  in  1901 
in  moldy  hazel  nut  meats.  It  de- 
velops quickly  in  beef  bouillon  where 
it  multiplies  by  budding  and  pro- 
duces ascs.  The  cells  are  very  elon- 
gated and  possess  a  double  wall  (Fig. 
138,  8  and  13).  In  old  cultures,  Fig.  138.  —  Nematospora  c&ryli. 

they    become    rOUnd    Or    OVal.      Bud-  I,    Ascospore  after   Disappearance  of    Cilium. 

...                                             1-11  2  to  6,  Germination  of  Ascospore;   7  to  8, 

dlllg    IS    always    accomplished    at    the  Ascs;    9  to  13,  Vegetative  Cells  in  Process  of 

\  Budding;   14  to  16,  Abnormal  Forms;   17  to 

poles   aS    in  the  yeastS  Of    DematlUm.  IS,  Stained    Ascospores   with    Nucleus   and 

JL,                     JIT-                                   j  Cilium  (after  Peglion). 

The  ascs  develop  plain  on  agar  and 

especially  on  slices  of  beet.     They  are  very  large  (65-70  jit  long  and 

6-8  M  wide).     They  possess  8  ascospores  placed  in  groups  of  four  in 

1  Peglion,   V.     Ueber  die   Nematospora  Coryli.     Notiz,   Rendie   della  Roy. 
Ac.  dei  Linei,  1897,  Cent.  Bakt.  7,  1901. 


NEMATOSPORA  LYCOPERSICI 


291 


each  half  of  the  cell.  (Fig.  138,  7  and  8.)  The  ascospores  are  very 
long:  they  have  the  shape  of  a  spindle  (2  to  3/x,  wide  and  38  to  40 /x 
long)  and  are  equipped  with  a  long  cilium  at  one  end.  During  ger- 
mination the  cilium  disappears  and  the  ascospore  takes  the  appear- 
ance of  a  short  cell  which  may  produce  buds  at  both  extremities. 
This  yeast  vegetates  only  on  a  solid  medium.  In  liquid  media,  it 
stops  budding  and  develops  only  a  sterile  mycelium.  To  this  fifth 
group  of  yeasts  seems  to  belong  a  species  found  by  Btitschli  in  a 
Nematode  Tylenchus  pellucidus. 


NEMATOSPORA  LYCOPERSICI.     Schneider1 

Schneider  has  recently  described  a  yeast  isolated  from  tomatoes 
secured  from  a  restaurant  in  Berkeley,  California.  The  proprietor 
stated  that  the  tomatoes  came 
from  the  South  Sea  islands. 
The  tomato  appeared  to  be 
normal  except  for  the  foci  of 
infection  which  were  depressed 
and  reddish  brown  in  color. 
Schneider  characterizes  this 
yeast  as  follows: 

"Asci  of  gametic  origin 
soon  becoming  free  from  as- 
sociated cells,  cylindrical  with 
rounded  ends,  60  to  70  jit  in 
length;  ascospores  in  two 
groups  of  four  spores  each, 
two-celled,  slender,  with 
pointed  ends,  slightly  ridged 
at  transverse  septum;  50  X 
4.5jii;  ascospores  liberated  by 
dissolution  of  the  ascus  wall 
and  held  together  somewhat  Fig.  138-A.  —  Nematospvra  Lycopersid.  Show- 
in  groups  of  4  by  motionless  ing  the  Shape  of  the  Cells  and  the  Formation 
a  «  a  «  «>  A  «™  of  Daughter  Cells  (after  Schneider), 

flagellae;   flagellae  50  to  100  ^ 

in  length;  arthrospores  of  non-gemetic  origin,  spherical  to  ampulliform, 
25 /i  in  diameter.  Two  other  cell  forms  also  found:  (l)  much  elon- 
gated filamentous  cells;  (2)  elliptical  or  ovoid  cells,  gametic  in  func- 
tion, new  cells  formed  in  bipolar  direction  by  apical  budding  and  also 

1  Schneider,  A.  A  parasitic  saccharomycete  of  the  tomato.  Phytopathology 
6  (1916)  395-399.  Further  note  on  a  parasitic  saccharomycete  of  the  tomato. 
Phytopathology,  7  (1917)  52-53. 


292 


FAMILY   OF  SACCHAROMYCETACEAE 


Fig.  138-B.  —  Arthro- 
spore  Formation  in 
Nematospora  Lyco- 


B,  (after  Schneider). 


by  apico-lateral  budding  at  cell  unions.  The  ellip- 
tical and  ovoid  cells  alone  are  gametic  in  func- 
tion." Apparently  this  yeast  is  a  parasite  on  the 
ripe  fruit  of  Lycopersicum  esculentum. 

Genus  XV.     Coccidiascus.     Chatton 

Budding  cells,  ascs  seem  to  be  derived  from 
an  isogamic  copulation  with  eight  ascospores. 

COCCIDIASCUS  LEGERI.     Chatton1 

This  yeast  was  observed  in  a  Muscide  Droso- 
phila  funebris.  The  yeast  infects  the  cells  of  the 
middle  intestine  and  lives  in  the  vacuoles  of  the 
cells  in  which  it  multiplies  by  ordinary  budding 
Budding  consti- 


and  also  by  ascs. 

tutes  the  intracellular  multiplication 
while  ascs  are  the  external  agents  of 
propagation.  The  parasite  possesses 
the  form  of  a  yeast  and  never  pro- 
duces a  mycelium.  Formation  of 
ascs  seems  to  be  preceded  by  an 
isogamic  copulation.  The  ascs  have 
the  shape  of  bananas.  They  contain 

8,1  •    i       i  f     1,   Vegetative  cells.      2,  Copulation.      3, 

aSCOSpOreS,     the     Special     Shape     OI         4,  Ascospores  Detached  from  Asc.   5-6, 

which  makes  this  yeast  resemble 
Monospora  cuspidata  and  Nematospora  coryli. 


Fig.  138-C.  —  Coccidiascus  Legeri. 

3,  Ascs. 
As- 


1  Chatton.     Coccidiascus  Legeri.  nov.  gen.  n.  sp.     Levure  ascospore  parasite 
de  cellule  intestinale  de  Drosophila  funebris.    Comp.  Rend.  Soc.  Biol.  75,  1913. 


CHAPTER  XI 
THE  NON-SACCHAROMYCETES   OR  DOUBTFUL  YEASTS 


T 


HIS  is  a  provisional  group  in  which  one  may  place  all  yeasts 
which  do  not  form  ascospores  and  whose  places  in  classifica- 
tion are  uncertain. 


Genus  Torula.     Turpin 

Under  the  name  Torula,1  Hansen  has  grouped  a  large  number  of 
asporogenic  species  often  capable  of  fermenting  sugars,  among  which 
are  some  which  do  not  form  a  scum  and  others  which  form  a  ring  around 
the  culture  flask  or  scum  without  the  interposition  of  air,  analogous 
to  those  which  are  produced  by  the  Saccharomycetes  of  the  third 
group.  The  greater  number  of  the  Torulae  possess  regular  spherical 
cells  and  contain  a  globule  of  fat  which  gives  them  a  special  charac- 
teristic. They  may,  then,  be  regarded  as  asporogenic  Torulaspora. 
But  some  are  oval  or  elongated  and  offer  the  appearance  of  ordinary 
yeasts.  A  certain  number  of  them  form  a  red,  black  or  brown  pig- 
ment. The  formation  of  these  pigments  seems  to  depend,  at  times, 
on  the  presence  of  silver  in  the  medium.  Light  sometimes  represses 
the  production  of  pigments.  The  investigations  of  Chapman  seem  to 
indicate  that  the  coloring  matter  in  red  torula  is  due  to  some  other 
substance  along  with  carrotene.  The  Torula  are  very  widespread  in 
nature.  They  are  encountered  in  the  soil,  breweries,  insects,  milk, 
in  secretions  of  trees,  etc. 

A.  TORULA  FROM  BREWERIES  AND  OTHER  SOURCES 

HANSEN'S  TORULA 

Hansen  2  has  described  a  number  of  Torula  which  we  shall  now 
consider. 

Torula  No.  I.  It  appears  in  beer  wort  as  isolated  cells  or  as 
colonies  formed  by  a  small  number  of  cells.  The  cells  are  1.5  to  4.5  jit 
in  diameter  and  often  have  a  large  vacuole  with  a  large  refractive 
body.  This  yeast  forms  a  small  amount  of  alcohol  in  beer  wort  and 
does  not  secrete  invertase. 

1  This  name  leads  to  confusion  for  it  is  also  applied  to  the  Mucedinaceae, 
very  different  from  the  yeasts   (genus  Torula  of  Persoon) ;    in  spite  of  this  the 
name  is  commonly  used. 

2  Hansen,  E.   C.     Sur  les  Torula  de  M.   Pasteur.     Comp.  Rend,   des  trav. 
du  lab.  de  Carlsberg,  2,  1888. 

293 


294    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

Torula  No.  2.     This  yeast  differs  from  the  preceding  by  its  larger 
cells,  from  3  to  8  jit  in  diameter,  and  granular  protoplasm. 

Torula  No.  3.  Morphologically  similar  to  the  pre- 
ceding yeast,  this  yeast  produces  7  to  8  per  cent  of 
alcohol  by  volume  in  beer  wort.  It  forms  a  foam  and 

Fig.    1  3  9.  —  sets  free  much   carbon   dioxide.    No  invertase   is   pro- 

Tontla  6  of  i         , 

Hansen.  duced* 

Growth    of  Torula    No.  4.     It  possesses  cells  from  2  to  6  ju  in 

Sediment  in  diameter.  It  inverts  saccharose  and  gives  a  little  more 
Beer  Wort  than  1  per  cent  of  alcohol  by  volume  in  beer  wort  with 
at  25°  the  formation  of  much  foam.  It 


C.    (after   does  not  ferment  maltose. 
Hansen). 


resembles  Torula  No.  1  in  the  shape  and  dimen- 

sions of  its  cells.     It  forms  a  homogeneous  dull  _. 

.   Fig.    140.  —  Torula  7  of 
gray  scum  on  beer  wort  at  the  temperature  of      Hansen.      Growth    of 

the  laboratory  and  in  other  liquids  containing       gells   «*    Sediment   in 

,  J  „     .     ,    .       T          T    ,  .  e  Beer    Wort    after   One 

10    per   cent  of   alcohol.     In   solutions   of  sac-       Day  at  25°  C.    (after 

charose,  it  forms  a  thin  scum.    It  inverts  sac-      Hansen). 
charoee  but  produces  only  traces  of  alcohol  in  sugar  solutions. 

Torula  No.  6.     It  possesses  spherical  or  oval  cells.    The  limits  of 
temperature  for  budding  are  4-6°  C.  and  36-37°  C.     (Fig.  139.)     This 

species  causes  an  apparent  fermentation 
in  beer  wort  and  yields  1.3  per  cent  of 
alcohol.     It  produces  no  fermentation  in 
maltose  solutions.     It  inverts  saccharose 
and  forms  5.1  to  6.2  per  cent  of  alcohol 
by   volume   in   yeast   water  with   added 
sugars  at  25°  C.  after  15  days;    after  two 
Fig.  141.  —  Torula  7  of  Hansen   months,    seven    per    cent    of    alcohol    is 
on  Scum  at  the  End  of  10  formed.     In  solutions  of  dextrose,  it  pro- 
Months  in  Wort  (after  Han-   ^^  ^   ^  ^  ^  Q£ 


volume. 

Torula  No.  7.  Found  in  the  soil  under  vines,  this  species  is 
made  up  of  ordinary  oval  cells  larger  than  those  in  the  preceding 
yeast.  (Fig.  140.)  The  oval  cells  are  irregularly  formed.  (Fig.  141.) 
The  temperature  limits  for  budding  are  0.5  and  38-39°  C.  This  species 
produces  1  per  cent  of  alcohol  by  volume  in  beer  wort.  It  does  not 
ferment  maltose  or  invert  saccharose.  In  yeast  water  with  dex- 
trose added  (10  or  15  per  cent)  it  yields  at  the  end  of  15  days,  4.5  per 
cent  of  alcohol  by  volume.  After  28  days,  it  produces  4.8  to  5.3  per 
cent  of  alcohol.  Hansen  thinks  that  this  species  like  the  preceding 
participates  in  the  fermentation  of  wine  and  cider. 


WILL'S  TORULA  295 

WILL'S  TORULA 

Will *  has  isolated  from  the  cooling  apparatus  and  from  the  air  in 
breweries,  etc.,  seventeen  forms  of  Torula  as  follows: 

Will's.  Torula  No.  1.  This  species  produces  a  thin,  white,  dull 
scum  and  a  very  evident  ring.  It  forms  a  very  evident  deposit  at 
the  bottom  of  the  culture  flask.  The  scum  and  ring  appear  at  the 
end  of  the  third  day.  The  thermal  death  point  of  this  yeast  in  beer 
wort  and  water  is  65°  C.  The  cells  are  very  small,  circular  in  shape 
like  the  typical  Torula.  A  few  elongated  and  sausage-shaped  cells 
may  also  be  found. 

Will's  Torula  No.  2.  The  scum  and  ring  look  like  those 
in  Will's  Torula  No.  1.  The  cells  are  oval  and  sometimes  very  elon- 
gated in  the  form  of  a  sausage.  This  species  perishes  at  60°  C.  in  must 
and  water. 

Will's  Torula  Nos.  3  and  4.  These  species  form  a  very  thick 
folded  scum,  of  a  whitish  yellow  color,  and  also  a  well-developed 
ring.  At  the  bottom  of  the  culture  flask  there  is  an  abundant  sedi- 
ment. The  scum  develops  rapidly  and  covers  the  surface  of  the  cul- 
ture three  days  after  inoculation.  The  cells  are  shaped  like  the  typical 
Torula.  These  two  species  are  almost  identical.  They  are  distin- 
guishable only  by  the  degree  of  changes  in  culture  media.  Both  are 
killed  at  60°  C.  in  must  and  water. 

Will's  Torula  No.  5.  This  species  is  most  often  round  or  oval. 
It  yields  a  very  thin  scum  of  small  islands  and  a  slight  ring.  The 
sediment  is  mucous  and  well  developed.  Under  certain  conditions 
this  yeast  gives  on  beer  wort  a  flowing  appearance.  The  cells  are 
killed  at  65°  C.  in  must  and  water. 

Will's  Torula  No.  7.  The  cells  are  spherical  with  a  spongy  in- 
terior and  a  very  distended  membrane.  An  abundant  deposit  is 
formed  at  the  bottom  of  the  culture  flask  and  on  the  surface  a  very 
mucous  scum.  In  cultures  which  are  a  little  old,  it  forms  a  thick  mu- 
cous sediment  filling  the  entire  volume  of  wort  which  is  changed  into 
a  compact  mass.  The  thermal  death  point  is  around  60°  C.  in  wort 
and  water. 

Will's  Torula  No.  8.  The  cells  are  large,  oval  and  often  elon- 
gated. A  well-developed  ring  is  produced,  a  feeble  scum  and  a  mucous 
irregularly  developed  sediment  at  the  bottom  of  the  culture  flask. 
The  thermal  death  point  in  beer  wort  and  water  is  60°  C. 

Will's  Torula  No.  9.  The  cells  are  very  small  and  oval  with 
some  elongated  to  the  shape  of  a  sausage.  A  feeble  ring  is  formed, 

1  Will,  H.  Beitrage  zur  Kenntniss  der  Sprosspilze  ohne  Sporenbildung. 
Zeit.  f.  d.  Ges.  Brauw.  26,  1903;  19,  1907;  Cent.  Bakt.  17,  1907;  21,  1908. 


296    NON-SACCHAROMYCETES   OR  DOUBTFUL  YEASTS 

a  well-developed  scum,  forming  small  islands,  and  an  abundant  de- 
posit. The  thermal  death  point  is  65°  C.  in  beer  wort  and  water. 

Will's  Torula  No.  10.  The  cells  of  this  species  are  similar  to 
those  of  No.  9.  A  powerful  ring  is  formed,  a  well-developed  scum 
and  a  rich  sediment.  The  thermal  death  point  is  about  60°  C. 

Will's  Torula  No.  11.  The  cells  are  small,  spherical  and  oval.  A 
ring  is  produced  with  a  folded  scum  of  a  white  color.  At  the  end  of  3 
days,  the  surface  of  the  culture  fluid  is  covered  with  this  scum  and 
at  the  bottom  of  the  flask  there  is  an  abundant  sediment.  This 
yeast  succumbs  at  65°  C.  in  must  and  water. 

Will's  Torula  No.  12.  This  yeast  is  composed  of  elongated,  or 
fusiform  cells  closely  resembling  those  of  Mycoderma.  There  are  also 
present  a  few  rounded  and  oval  cells.  A  strong  scum  much  folded 
of  a  light  yellow  or  brown  color  is  produced.  It  may  become  a  pale 
red  after  three  months.  This  scum  resembles  that  of  Mycoderma  very 
much.  It  appears  soon  as  little  floating  islands  which  look  like 
fat  globules.  The  thermal  death  point  in  beer  wort  and  in  water  is 
60°  C. 

Will's  Torula  No.  15.  The  cells  have  different  shapes,  most 
often  elongated,  oval  or  shaped  like  a  sausage.  The  scum  is  well  de- 
veloped, very  much  folded  and  resembles  that  of  Mycoderma.  It  has 
a  white,  slightly  yellow,  color.  It  begins  to  appear  at  the  end  of  three 
days.  The  cells  succumb  at  65°  C.  in  must  and  in  water. 

Will's  Torula  No.  16.  It  is  made  up  of  spherical  or  oval  cells 
mixed  with  long  cells.  The  scum  is  well  developed,  compact  and  of  a 
yellowish  white  color.  A  mediocre  amount  of  sediment  is  formed. 
The  scum  appears  on  the  third  day.  The  thermal  death  point  in 
must  and  water  is  60°  C. 

Will's  Torula  No.  17.  The  cells  are  small,  quite  round  and  pos- 
sess the  typical  Torula  shape.  The  scum  is  moderately  developed, 
compact  and  white.  The  thermal  death  point  in  must  and  water  is 
60°  C. 

The  greater  number  of  these  Torula  are  not  distinct  species. 
More  often  they  are  of  simple  varieties  or  different  races.  All  grow 
at  low  temperatures  on  beer  wort.  The  minimum  temperature  for 
budding  is  5-6°  C.  The  optimum  temperature  is  30-35°  C.  Almost 
all  of  them  impart  to  beer  wort  an  agreeable  fruity  odor.  Species 
Nos.  7  and  8,  however,  produce  a  disagreeable  odor. 

Will l  has  recently  described  a  number  of  other  species  found  in 
and  about  breweries.  They  have  been  characterized  as  follows: 

1  Will,  H.  Beitrage  zur  Kenntniss  der  Sprosspilze  ohne  Sporenbildimg  welche 
in  Brauereibetrieben  und  in  deren  Umgebung  vorkommen.  Cent.  Bakt.  Abt.  2, 
46,  1916. 


WILL'S  TORULA  297 

Torula  Nos.  3  and  4.  Cells  spherical  (3-4  ju).  The  giant  colonies 
are  thick  with  a  flat  edge  which  is  sinuous.  It  ferments  dextrose, 
levulose,  galactose  and  raffinose.  A  scum  is  formed  on  liquid  media, 
dry  and  white  at  first,  later  changing  to  a  brownish  yellow.  The  tem- 
perature limits  for  growth  on  wort  are  0.5  and  33°  C.  It  was  found  in 
the  air  about  breweries. 

Torula  No.  11.  The  cells  are  spherical  or  ellipsoidal  (3-4 /z). 
A  scum  is  formed  in  liquid  media  which  is  dry  and  folded.  It  is  of  a 
gray  color.  The  giant  colony  exists  in  the  form  of  a  flat  layer  with 
a  united  edge.  It  ferments  dextrose,  levulose,  galactose  and  sac- 
charose. The  temperature  limits  for  growth  on  beer  wort  are  0.5 
and  33°  C.  It  was  isolated  from  wort. 

Torula  No.  17.  The  cells  are  spherical  and  sometimes  ellipsoidal. 
(About  3/z  in  diameter.)  A  thin  dry  scum  is  formed  in  liquid  media 
of  a  flat  white  or  yellow  color;  it  is  slightly  folded  and  grips  the  sides 
of  the  culture  flask.  The  giant  colonies  are  round.  It  ferments  dex- 
trose, levulose,  galactose,  saccharose,  lactose  and  raffinose.  It  was 
isolated  from  brewing  water. 

Torula  No.  6.  The  cells  are  spherical,  sometimes  ellipsoidal.  They 
develop  in  liquid  media,  especially  in  the  form  of  a  sediment.  A  very 
thin  scum  appears  quickly.  The  giant  colonies  are  like  those  of 
Torula  No.  2.  It  ferments  dextrose,  levulose,  galactose,  saccharose 
and  maltose.  The  temperature  limits  for  growth  on  beer  wort  are  0.5 
and  30°  C. 

Torula  No.  5.  The  cells  are  spherical,  ellipsoidal  or  elongated. 
A  scum  and  ring  are  formed  on  liquid  media.  The  scum  is  colorless, 
moist  and  thin.  Dextrose,  levulose,  saccharose,  galactose,  maltose 
and  raffinose  are  fermented.  The  temperature  limits  for  growth  are 
0.5  and  35°  C.  It  was  isolated  from  wort. 

Torula  No.  7.  The  cells  are  spherical  and  at  the  beginning 
develop  at  the  bottom  of  the  liquid.  A  ring  and  a  scum  are  finally 
formed.  The  ring  is  solidly  adherent  and  of  a  gelatinous  consistency. 
The  scum  is  well  developed,  mucous  and,  at  first,  uncolored.  The 
giant  colony  is  a  reddish  brown  at  first,  finally  a  brown.  It  ferments 
dextrose,  levulose,  galactose,  saccharose,  maltose,  lactose,  raffinose 
and  arabinose.  The  temperature  limits  are  2°  C.  and  30°  C.  It  was 
isolated  from  the  air. 

Torula  No.  8.  The  cells  are  spherical  or  ellipsoidal,  slightly 
pointed.  They  become  elongated  at  low  temperatures  and  may  reach 
5ju.  In  liquid  cultures  there  is  feeble  development  as  a  sediment 
but  a  scum  and  a  mucilaginous  ring  are  formed.  The  giant  colonies 
are  a  deep  brown.  It  ferments  dextrose,  levulose,  galactose,  maltose, 
lactose,  raffinose  and  arabinose.  The  temperature  limits  are  0.5  and 
35°  C. 


298    NON-SACCHAROMYCETES  OR  DOUBTFUL   YEASTS 

Torula  No.  9..  The  cells  are  ellipsoidal,  sometimes  in  the  shape 
of  lemons.  There  is  a  ring  formed  but  no  scum.  The  giant  colonies 
are  flat  and  transparent.  It  ferments  dextrose,  levulose,  galactose, 
maltose,  lactose,  raffinose  and  arabinose.  The  temperature  limits 
are  0.5  and  40°.  It  was  isolated  from  brewing  water. 

Torula  No.  1.  The  cells  are  spherical  or  ellipsoidal.  On  liquids 
the  development  is  rapid  with  a  superficial  vegetation,  at  first  in  the 
form  of  a  ring,  and  later  in  a  flat  and  white  scum.  It  ferments  dex- 
trose, levulose,  saccharose,  maltose,  lactose,  raffinose  and  arabinose. 
The  temperature  limits  are  2°  and  40°  C.  It  was  isolated  from  brew- 
ing water. 

Torula  No.  2.  The  shape  and  size  of  the  cells  of  this  yeast  are 
variable,  in  general,  spherical  or  ellipsoidal.  There  are  many  giant 
cells  in  the  cultures.  On  liquid  media  it  develops  like  the  former 
yeast.  The  giant  colonies  have  a  sort  of  crater  in  their  centers  and  a 
thin  marginal  part.  It  ferments  dextrose,  levulose,  galactose,  sac- 
charose, maltose,  raffinose  and  arabinose.  The  temperature  limits 
are  0.5  and  39°  C. 

Torula  No.  10.  The  cells  are  ellipsoidal  or  elongated.  On  liquid 
media,  the  development  is  slow  in  the  beginning  with  a  sediment al 
growth  but  finally  a  white  or  rose  scum  is  formed.  Gelatin  is  rapidly 
liquefied.  The  yeast  ferments  dextrose,  levulose,  galactose,  saccharose, 
maltose,  lactose  and  arabinose.  The  temperature  limits  are  2°  C.  and 
35°  C. 

Torula  No.  15.  The  cells  are  spherical  or  ellipsoidal.  It  develops 
slowly  on  liquid  media  with  a  superficial  vegetation.  The  scum  is 
dull  white  in  the  beginning,  then  yellow.  It  ferments  dextrose,  levulose, 
galactose,  saccharose,  maltose,  lactose,  raffinose  and  arabinose.  The 
temperature  limits  are  0.5  and  35°  C. 

Torula  No.  16.  The  cells  are  spherical  and  sometimes  ellipsoidal. 
They  grow  rapidly  on  liquid  media.  A  yellowish  brown  scum  is 
formed.  It  ferments  dextrose,  levulose,  galactose,  saccharose,  malt- 
ose, lactose,  raffinose  and  arabinose.  The  temperature  limits  are 
0.5°  C.  and  30°  C. 

Torula  No.  12.  The  cells  have  a  very  variable  appearance.  They 
are  either  round  or  ellipsoidal  and  small.  It  develops  on  liquid  media, 
almost  always  in  the  form  of  a  scum,  at  first  white,  later  becoming  yel- 
low. The  giant  colonies  are  reddish  in  their  centers  and  white  around 
the  periphery.  It  ferments  dextrose  and  levulose  actively  and  malt- 
ose, galactose  and  saccharose  feebly.  The  temperature  limits  are  2° 
and  35°  C. 


TORULA  BRETTANOMYCES  299 

TORULA  OF  LINDNER  AND  MEISSNER 

Lindner  l  has  described  two  Torula  in  detail  from  the  collection  at 
Berlin.  They  are  Torula  Nos.  63  and  64.  Their  cells  often  reach  the 
size  of  beer  yeast  and  present  a  very  granular  protoplasm.  The  first 
of  these  two  species  forms  a  cartilaginous  scum  on  beer  wort,  difficult 
to  crush  under  the  cover  slip  with  a  moist  and  transparent  appear- 
ance. On  wort  gelatin  in  streaks,  it  produces  a  mucous  deposit, 
sometimes  cartilaginous.  Under  the  same  conditions,  the  second 
variety  produces  a  mucous  sediment.  The  cells  of  both  species  rarely 
contain  fat  globules  and  possess  a  thick  membrane,  the  exterior  mem- 
brane of  which  shows  a  tendency  to  detach  itself.  Eleven  forms  of 
Torula  have  been  isolated  by  Meissner.2  They  cause  a  defect  in  wine 
in  which  it  becomes  thick  and  greasy. 

TORULA  NOVAE  CARLSBERGIAE.    Gronlund3 

This  species  has  cells  of  various  shapes  and  gives  a  disagreeable 
taste  to  beer  wort.  According  to  Schjerning,  it  inverts  saccharose 
and  sets  up  an  alcoholic  fermentation  in  solutions  of  saccharose,  dex- 
trose and  maltose.  In  beer  wort,  it  is  able  to  reproduce  about  4.7 
per  cent  of  alcohol  by  volume. 


TORULA  BRETTANOMYCES.    Clausen 

Under  this  name,  Clausen 4  described  a  special  group  of  Torula 
which  produced  a  secondary  fermentation  in  English  beers.  It  differs 
from  other  Torula  in  that,  if  a  preliminary  fermentation  is  carried  on 
by  Saccharomyces,  it  will  multiply  and  ferment  the  remaining  sugar. 
It  forms  acids  which  combine  with  the  alcohols  to  form  aromatic 
substances  giving  the  beer  a  special  flavor  and  aroma.  Schionning  5 
has  studied  this  group.  The  Torula  which  make  up  this  group  fer- 
ment maltose  actively.  In  beer  wort  with  about  10  per  cent  saccha- 
rose, they  form  about  10  per  cent  of  alcohol  by  volume  but  the  fer- 

1  Lindner,  P.    See  reference  for  Willia  belgica. 

2  Meissner,  R.     Studien  iiber  das  Zahnwerden  von  Most  u.  Wein.     Landw. 
Jahrb.  27,  1898. 

3  Gronlund,  Ch.     En   ny  Torula-Art  og   to  nye  Saccharomyces  Arter.     Vi- 
densk.  medd.  fra  den  Natur.     Foren.     Copenhagen,  1892. 

4  Clausen,  H.     Occurrence  of  Brettanomyces  in  American  Lagerbeer.     Amer. 
Brewing  Rev.  19,  1905. 

6  Schionning,  H.  On  Torula  in  English  Beer  Manufacture.  Comp.  Rend, 
trav.  lab.  de  Carlsberg,  7,  1908. 


300    NON-SACCHAROMYCETES   OR  DOUBTFUL   YEASTS 

mentation  is  accomplished  slowly  and  only  ends  after  eight  months 
at  20°  C.  They  ferment  sucrose  without  giving  products  which  reduce 
Fehling's  solution,  although  an  inversion  of  the  saccharose  is  brought 
about.  According  to  Schionning,  these  types  may  be  distinguished 
in  these  ways.  Torula  A  may  be  distinguished  from  Torula  B  by  its 
action  towards  sugars  and  also  by  the  temperature  limits  for  budding. 

Torula  A.  The  cells  are  ellipsoidal  while  some  are  sausage  shaped 
or  in  the  form  of  a  mycelium.  Others  present  odd,  irregular  shapes. 
The  size  of  the  cells  is  variable.  Giant  cells  are  found  with  a  very 
refractive  protoplasm,  showing  vacuoles  scarcely  visible  in  which  a 
mobile  granule  is  often  found.  The  temperature  limits  for  budding 
are  5-7°  C.  and  40-40.5°  C.  The  races  of  type  A  produce,  in  old  liquid 
cultures,  a  loose  scum  in  which  the  cells  are  filamentous,  resembling  a 
mycelium.  It  ferments  dextrose,  levulose,  saccharose  and  maltose 
(saccharose  more  actively  and  maltose  less  actively  than  type  B). 
It  has  no  action  on  lactose  and  dextrine. 

Torula  B.  The  cells  resemble  somewhat  those  of  Torula  A.  They 
are  often  sausage  shaped.  Long  mycelial  structures  are  found  among 
them.  The  temperature  limits  for  budding  are  3-4°  C.  and  39-39.5°  C. 
In  old  liquid  cultures,  the  races  of  type  B  form  a  scum  identical  with 
that  of  Torula  A.  They  ferment  saccharose,  maltose,  dextrose,  levu- 
lose and  lactose  but  do  not  act  on  dextrose. 

BEIJERINCK'S  TORULA 

Beijerinck  l  has  isolated  three  yeasts  which  do  not  sporulate  and 
which  ought  to  be  classed  among  the  Torula.  They  are: 

Saccharomyces  fragrans,  Beijerinck.  According  to  Beijerinck,  this 
yeast  is  a  contamination  in  compressed  yeast.  It  has  been  insuffi- 
ciently described. 

Saccharomyces  muciparus,  Beijerinck.  Related  to  the  preceding 
species,  this  is  also  incompletely  known.  It  is  characterized  by  a 
very  evident  polymorphism.  In  old  cultures,  it  presents  filamentous 
and  yeast  forms. 

Saccharomyces  curvatus,  Beijerinck.  This  yeast  was  isolated  by 
Beijerinck  from  an  impure  culture  of  S.  muciparus.  It  seems  to 
correspond  to  the  cheesy  yeast  described  by  Pasteur.  The  shape  is 
much  like  that  of  Saccharomyces  Pastorianus.  It  is  curved,  however, 
whence  the  name  S.  curvatus.  This  yeast  has  the  characteristic,  as 
Pasteur  found,  of  not  being  broken  up  in  water.  It  settles  to  the 
bottom  of  the  culture  flask  as  a  curd  leaving  the  supernatant  liquid 

1  Beijerinck,  M.  W.  Die  Erscheinung  des  Flokenbildung  oder  Agglutination 
bei  Alkoholhefen.  Cent.  Bakt.  20,  1908. 


SACCHAROMYCES   SPEC  301 

almost  clear.     According  to  Beijerinck,   this  yeast  seems  to  possess 
the  ability  of  agglutinating  itself. 


TORULA  COLLICULOSA.     Hartmann 

This  species  was  isolated  from  a  sample  of  yeasts  secured  from 
Java  by  Hartmann.1  It  was  associated  with  Mucor  amylomyces.  It 
presents  the  appearance  of  ordinary  Torula.  Its  cells  are  spherical 
and  contain  a  fat  globule.  Its  ordinary  dimensions  are  3. 5  IJL  but 
they  may  vary  between  1.7  and  9.7  JJL.  Budding  is  accomplished  on 
all  sides  of  the  cell.  Frequently  two  or  three  buds  appear  simul- 
taneously. The  cells  are  often  united  in  chains  of  two  or  three,  the 
mother  cell  being  much  larger  than  the  daughter  cell.  The  optimum 
temperature  for  budding  on  wort  agar  is  situated  between  25°  C.  and 
30°  C.  Budding  stops  at  7°  C.  and  45°  C. 

The  giant  colonies  obtained  on  beer  wort  agar  have  a  character- 
istic aspect.  They  form  a  prominent  wart.  On  beer  wort  gelatin, 
the  colonies  develop  well  and  liquefy  the  gelatin  after  eight  weeks. 
Cultivated  in  beer  wort  at  25°  to  30°  C.,  Torula  colliculosa  produce, 
at  the  end  of  24  hours,  a  powdery  white  sediment.  After  40  hours  the 
liquid  clarifies  itself.  Depending  on  their  age,  the  cells  act  differently 
on  maltose.  The  oldest  cells  are  able  to  ferment  this  sugar  while  the 
young  ones  have  no  action.  This  species  ferments  dextrose,  raffinose 
and  saccharose. 


SACCHAROMYCES    SPEC.     Saito 

Isolated  from  "  koji "  by  Saito,2  this  yeast  forms  on  decoction  of 
"  koji  "  or  on  beer  wort  globular  or  oval  cells,  without  color,  about 
4  to  7  jit  in  diameter.  The  cells  are  usually  isolated  and  show  one 
or  many  vacuoles  and  a  finely  granular  protoplasm.  In  old  cultures 
they  often  take  the  form  of  a  sausage.  On  gelatin  plates,  the  colonies 
first  appear  as  little  round  points,  later  changing  to  a  white  mass  in 
the  shape  of  a  dome  with  an  uneven  surface.  On  the  surface,  they 
appear  as  numerous  spots  made  up  of  elliptical  cells.  This  species 
seems  to  be  related  to  Saccharomyces  awamori.  It  ferments  dextrose, 
levulose,  d-galactose,  saccharose,  maltose,  melibiose,  raffinose  and  a- 
methylglucoside.  It  has  no  action  on  inuline  or  lactose.  In  wort 
after  20  days,  it  produces  5.99  per  cent  of  alcohol  by  volume. 

1  Hartmann,  M.     Eine  rassespaltige  Torula- Art  welche  nur  zeitweise  Maltos 
zu  vergaren  vermag.    Wochenschr.  f.  Brau.  20,  1903. 

2  Saito,    K.      Notes   on   Formosan   fermentation   organisms.      The    Botanical 
Magazine,  15,  Nos.  21  and  52,  1902. 


302    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 


SACCHAROMYCES  AWAMORI.     Inui 

This  species  was  found  by  Inui l  who  isolated  it  from  foamy  wine 
of  the  Japanese  called  awamori.  It  produces  on  gelatin  an  irregular 
colony.  It  is  able  to  stand  three  hours'  exposure  at  50°  C.  It  de- 
velops in  liquids  containing  8  per  cent  of  alcohol  but  growth  is  stopped 
by  20  per  cent.  In  beer  wort,  it  endures  6  per  cent  of  alcohol. 


DE  KRUYFF'S  TORULA 

De  Kruyff 2  isolated,  from  the  soil  and  living  and  dead  leaves  in 
Java  along  with  Zyg.  javanicus,  seven  species  of  yeasts  under  the  name 
of  Saccharomyces  javanicus  1-7.  These  are  ellipsoidal  or  round  yeasts 
all  of  which,  with  the  exception  of  one,  ferment  saccharose,  dextrose 
and  maltose.  As  no  sporulation  has  been  described  for  them  they  may 
be  regarded  as  Torula. 

TORULA  OF  PEARCE  AND  BARKER 

Pearce  and  Barker  3  have  isolated  a  series  of  Torula  from  cider  in 
England. 

Yeast  C.  This  is  a  yeast  with  small,  spherical  or  oval  cells 
(4.5  X  3.7  ju).  The  maximum  temperature  for  budding  is  37-37.5°  C. 
On  gelatin  plates,  it  forms  damp,  transparent,  round  colonies.  On 
streaks,  it  gives  a  moist  vegetation  with  indented  rugose  edges. 

Yeast  D.  The  cells  of  this  yeast  are  generally  oval  (4.5  X  3.7/i), 
sometimes  elongated  and  shaped  like  a  sausage.  The  maximum  tem- 
perature for  budding  is  32°  C.  The  cultures  on  gelatin  plates  are 
round  and  dry  with  a  tendency  to  flatten  onto  the  gelatin.  The 
edges  are  irregular  and  festooned.  On  streaks  the  growth  becomes 
slightly  fringed  at  the  edges.  This  species  forms  a  scum  on  all  sugar 
media  but  does  not  cause  a  fermentation. 

Yeast  E.  The  cells  are  oval  shaped  (11.9-6.8  X  3.4  ju).  The 
maximum  temperature  for  budding  is  situated  near  38°  C.  This 
species  forms  a  well-developed  scum  on  solutions  of  maltose.  It- 
does  not  produce  fermentation. 

1  Inui,  T.  Untersuchungen  uber  die  niederen  Organismen  welche  sich  bei 
der  Zubereitung  des  alkoholischen  Getranks  Awamori  beteiligen.  Jour,  of  the 
College  of  Sciences.  Univ.  Tokyo.  15,  1901. 

-  De  Kruyff,  E.  Untersuchungen  iiber  auf  Java  einheimische  Hefenarten. 
Cent.  Bakt.  21,  1908. 

3  Pearce,  B.,  and  Barker,  P.  The  yeast  flora  of  bottled  ciders.  The  Journal 
of  Agricultural  Science,  3,  1908, 


SACCHAROMYCES  BRASSICAE  III.  303 

Yeast  J.  The  cells  of  this  yeast  are  oval  and  spherical  (8.5- 
6.8x3.4jit).  The  maximum  temperature  for  budding  is  situated 
between  30  and  32°  C.  Colonies  on  wort  gelatin  in  plates  are  round, 
moist  and  partly  transparent.  On  streaks,  the  growth  is  flat  and 
spreading.  This  species  ferments  dextrose,  levulose  and  saccharose. 

Yeast  L.  This  species  has  small  cells  shaped  like  a  sausage 
(12-6.8  X  2.7 /A).  The  maximum  temperature  for  budding  is  about 
38°  C.  Colonies  on  wort  gelatin  in  plates  are  spherical  with  thin 
edges.  On  streak  cultures  vegetation  is  compact.  This  species  fer- 
ments dextrose  and  maltose. 

Yeast  M.  The  cells  are  oval  (8-6.8  X  5 /z).  Sometimes  they 
are  sausage  shaped.  The  maximum  temperature  for  budding  is  situ- 
ated between  35  and  28°  C.  On  wort  gelatin  in  plates,  the  colonies 
are  dry,  spherical  and  with  a  solid  appearance.  Gelatin  is  liquefied 
after  a  certain  time.  This  species  ferments  dextrose,  levulose,  maltose 
and  saccharose. 

SACCHAROMYCES  BRASSICAE    I.  Wehmer 

Isolated  by  Wehmer,1  from  fermentation  of  sourkraut,  this  species 
presents  spherical  or  elongated  cells  closely  resembling  those  of  S. 
cerevisiae  but  smaller  (4-6  X  5 /z).  The  development  on  agar  and 
gelatin  with  a  decoction  of  kraut,  in  stab  and  streak,  gives  firm, 
grayish  white,  slightly  raised  colonies.  The  sediment  in  a  fermenting 
liquid  forms  a  grayish  white  mass  from  which  escape  bubbles  of  gas. 
This  yeast  produces  an  active  fermentation  in  decoctions  of  kraut, 
especially  that  to  which  dextrose  is  added.  It  ferments  beer  wort. 

SACCHAROMYCES  BRASSICAE  II.    Wehmer 

This  yeast  was  isolated  by  Wehmer  from  the  same  source  as  the 
preceding  one.  The  cells  are  almost  spherical  and  do  not  exceed  3.6 
to  4.8  ju,  in  diameter;  often  they  are  much  smaller.  They  possess  very 
refractive  granules  in  their  vacuoles.  This  species  has  the  same  ap- 
pearance on  gelatin.  In  a  decoction  of  kraut  in  dextrose  solutions 
and  in  beer  wort,  it  produces  a  fermentation. 

SACCHAROMYCES  BRASSICAE  III.    Wehmer 

Isolated  also  from  sourkraut,  this  species  possesses  ellipsoidal 
cells,  slightly  elongated  and  quite  small  (7  X  4ju  in  diameter).  The 
appearance  of  the  cultures  on  gelatin  is  quite  different  from  that  of  the 
two  preceding  yeasts.  This  yeast  produces  an  alcoholic  fermentation. 

1  Wehmer,  C.  Untersuchungen  tiber  Sauerkrautgarung.  Cent.  Bakt.  2, 
1905. 


304    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

TORULA  HOLMII.     (Holm)   Jorgensen l 
Syn.:    TORULA  A.     Holm 

This  yeast  was  isolated  by  Holm  in  Jorgensen's  laboratory.  The 
growth  in  the  sediment  of  young  cultures  contains  small,  oval  cells. 
Along  with  these  are  found  large  cells,  oval  or  round.  The  length  of 
the  cells  varies  from  3.5  to  5.5/x  and  their  width  from  1.4  to  2.1/z. 
In  must  this  species  produces  a  feeble  fermentation  after  which  the  al- 
cohol content  may  reach  0.32  per  cent  by  volume.  It  inverts  sac- 
charose and  raffinose  and  ferments  the  invert  sugar.  However,  it 
has  no  action  on  maltose,  dextrose  and  dextrine.  At  the  end  of  three 
to  five  days  at  25°  C.,  it  forms  a  scum  on  beer  wort,  the  cells  of  which 
are  round  or  oval.  In  yeast  water  to  which  dextrose  has  been  added 
the  cells  of  the  scum  look  like  those  of  S.  Pastorianus.  They  may  also 
be  irregular.  The  surface  colonies  on  gelatin  with  10  per  cent  wort 
are  round,  white,  shiny,  slightly  bulged  and  with  entire  edge. 

TORULA  THERMANTITONUM.    Johnson 

Discovered  on  the  leaves  of  the  Eucalyptus  and  studied  by  John- 
son2 and  Hare,  this  species  possesses  cells  of  small  size  which  are 
egg  shaped.  It  is  of  special  interest  on  account  of  an  abnormal  re- 
sistance to  high  temperatures.  The  temperature  limits  for  budding 
are  situated  in  the  vicinity  of  10°  C.  and  84°  C.  The  optimum  is  be- 
tween 40  and  44°  C.  According  to  Lindner,  it  is  a  brewery  yeast  which 
induces  a  bottom  fermentation  and  a  good  clarification.  It  ferments 
dextrose,  levulose,  saccharose,  maltose,  dextrine,  d-mannose,  d-galac- 
tose,  raffinose,  a-methylglucosides,  xylose  and  inuline  but  has  no  ac- 
tion on  lactose  and  melibiose. 

TORULA  FROM  VACCINE  PULP.    Lesieur  and  Mangini 

Lesieur  and  Mangini  have  found  the  presence  of  yeasts  in  samples 
of  vaccine  pulp.  These  yeasts  belong  to  the  genera  Mycoderma  and 
Torula. 

Torula  I.  The  cells  are  round,  more  often  spherical.  On  wort  at 
25°  C. ,  the  yeast  causes  a  cloudiness  after  40  hours,  and  very  slight 
scum  formation.  After  five  days,  there  is  visible  scum  and  sedimen- 
tal  growth  at  the  bottom  of  the  container.  The  yeast  grows  a  little 

1  Jorgensen,    A.       Die    Mikroorganismen    der    Garungsindustrie,    Berlin,   5th 
Edition,  Paul  Parey,  1909. 

2  Johnson,    G.     Saccharomyces   Journal   of   the   Institute   of    Brewing,     11, 
1905. 


WEHMER'S  TORULA  305 

at  14°  C.  The  optimum  temperature  for  growth  seems  to  be  situated 
between  25°  C.  and  37°  C.  At  37°  C.  the  yeast  vegetates  but  stops  at 
40°  C.  This  yeast  ferments  levulose.  The  inoculation  of  a  guinea  pig 
gave  negative  results. 

Torula  II.  The  cells  are  variable  in  shape,  either  spherical  or 
oval  (5-5. 5 /z,  in  diameter).  On  Raulin's  gelatin,  the  cells  are  round 
and  there  are  rudiments  of  a  mycelium.  On  liquid  media,  there  is 
a  sediment  and  after  10  to  12  days  a  slight  ring 
appears.  Growth  begins  at  14°  C. ;  the  optimum 
temperature  is  between  19  and  20°  C. 

Torula  III.  The  cells  are  ordinarily  spheri- 
cal, rarely  oval  (4-6. 5  ju).  On  potato,  the  cells 
are  united  into  groups  of  2,  3,  or  4  individuals,  Fig.  141-A.  —  Twulalll 
rarely  more.  There  is  a  gelatinous  substance  ^?m  .Vaccine  (after 
resembling  a  zoogloea.  On  liquid  media  an 

abundant  sediment  and  a  white  scum  are  formed  after  11  days. 
This  yeast  develops  at  14°  C.  The  maximum  temperature  for  bud- 
ding seems  to  be  situated  between  25°  and  37°  C.  The  species  is  able 
to  grow  at  45°  C.  /There  is  no  fermentation  and  it  is  non-pathogenic. 

Olsen-Sopp  have  isolated  from  Taette  along  with  Saccharomyces 
taette  major  and  minor  some  Torula  which  form  small  round  or  oval 
colonies  which  are  distinguishable  from  each  other  by  their  action 
toward  sugars. 

TORULA  OF  ROSE 

Rose  l  has  isolated  two  types  of  Torula  from  the  mucous  secretions 
of  oaks.  Both  have  round  cells  (5/z  in  diameter)  and  act  as  bottom 
yeasts.  One,  Torula  A,  ferments  dextrose,  trehalose,  ramnose  inu- 
line,  a-and  /3-glucosides  and  possibly  melibiose.  The  other,  Torula 
B,  acts  like  the  Torula  A  and  differs  only  by  the  fact  that  it  does 
not  act  on  inuline  and  melibiose. 


WEHMER'S  TORULA 

Wehmer2  has  isolated  from  pickle  brine  a  small  round  Torula 
which  vegetates  in  nutrient  solutions  containing  10  to  15  per  cent  of 
salt.  In  solutions  of  21  per  cent  of  salt  it  remains  capable  of  develop- 
ing for  months.  It  seems  to  originate  from  sea  water  or  the  herring. 

1  Rose,   L.     Beitrage  zur  Kenntniss  der  Organismen  in  Eichenschleimfluss. 
Inaugural  dissertation  at  the  University  of  Berlin.     June  1910. 

2  Wehmer,  C.     Zur  Bakter.  und  Chemie  der  Heringslake.     Cent.  Bakt.  3, 
1897. 


306    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 


TORULA  SP.    Saito1 

This  yeast  possesses  spherical  cells  with  resistant  walls  with  a 
number  of  fat  globules  in  the  protoplasm.  The  cells  form  a  mucilag- 
inous sediment.  On  a  fat  substrate  they  become  oval  or  elongated. 
The  giant  colonies  give  a  mucilaginous  stratification  of  a  yellow  color. 
In  wort  or  decoction  of  "koji,"  this  yeast  causes  a  feeble  escape  of 
gas;  at  first,  there  is  a  cloudiness  produced,  later,  the  formation  of  a 
united  scum.  This  species  is  closely  related  to  the  preceding  one  and 
possibly  identical. 

HOYE'S  TORULA 

Hoye2  found  many  species  of  Torula  in  solutions  to  which  salt 
had  been  added.  These  will  be  described. 

Salt  Yeast  B,  Hoye.  This  is  a  yeast  with  round  or  oval  cells, 
the  shape  varying  somewhat  with  the  concentration  of  salt.  When 
15  per  cent  of  salt  is  added,  the  cells  become  more  oval  than  with 
lesser  amounts.  With  20  per  cent  of  salt  the  cells  become  more  pointed, 
resembling  the  tubercles  of  beets.  The  membranes  of  the  cells  often 
have  short  points  to  the  number  of  two  or  three  on  each  cell.  Fre- 
quently the  cells  are  joined  together  by  means  of  these  points.  This 
yeast  does  not  grow  in  potato  wort. 

Salt  Yeast,  Hoye.  In  fish  bouillon  with  15  per  cent  of  salt  added, 
this  species  possesses  cells  of  various  shapes.  The  structure  is  often 
moniliform.  In  25  per  cent  of  salt,  the  cells  are  oval  and  located  in 
chains.  In  35  per  cent  of  salt,  they  become  round.  This  yeast  does 
not  develop  in  potato  wort.  Schultz  has  mentioned  yeasts  of  the 
ellipsoideus  type  from  brines  used  to  preserve  legumes. 

Recently,  Coupin  has  found  in  sea  water  a  Torula  which  does  not 
grow  in  media  unless  salt  is  added  to  it.  Kita  has  found  two  species 
of  Torula  which  develop  in  beer  wort  with  20  per  cent  of  added  salt. 
They  grow  easily  in  solutions  with  maltose  but  not  in  those  which 
contain  dextrose. 

1  Saito,    K.     Mikrobiol.    Stud,   iiber   die   Soya-Bereitung.     Cent.    Bakt.    17, 
1906. 

2  Hoye,    K.      Recherches   sur   la   moisissure   de   Bacalao   et   quelques   autres 
Mikroorganismes  halophiles.    Bergens.  Museum  Aarbog.    No.  12.     1906. 


DuCLAUX'S  TORULA  307 

B.     TORULA  FROM  MILK 

TORULA  KEPHIR.     Heinze  and  Cohn1 

Syn.:    SACCHAROMYCES  KEPHIR.    Beijerinck 

Found  by  Beijerinck  2  in  kephir,  this  yeast  possesses  cells  of  vari- 
able shape,  generally  elongated,  developing  on  nutrient  gelatin  with 
slightly  winding  colonies  and  not  liquefying  the  gelatin.  According  to 
Beijerinck  it  secretes  lactase.  Schurmans-Stekhoven,  however,  have 
not  been  able  to  confirm  this  fact.  It  inverts  saccharose  but  has  no 
action  on  maltose. 

SACCHAROMYCES  TYROCOLA.    Beijerinck 

This  species  was  isolated  by  Beijerinck 3  from  Eidam  cheese.  It  is 
a  yeast  with  small  cells,  round,  giving  snow-white  colonies  on  gelatin. 
As  with  the  preceding  species,  it  inverts  lactose  and  saccharose  but 
does  not  act  on  maltose. 

FREUDENREICH'S  KEPHIR  YEAST 

This  species  was  found  by  Freudenreich 4  in  various  samples  of 
kephir.  It  gives  a  vigorous  growth  and  feeble  fermentation  in  beer 
wort  but  does  not  seem  to  cause  any  fermentation  in  milk.  The 
growth  consists  of  oval  cells  (3.5/z  in  length  and  2-3  /*  in  width). 
It  does  not  form  a  scum.  The  optimum  temperature  for  budding  is 
22°  C.  According  to  Freudenreich,  this  species  is  able  by  a  symbiosis 
with  Dispora  causica  and  Streptococcus  a  and  |3  to  bring  about  a  fer- 
mentation in  milk.  It  does  not  act  on  lactose  when  it  is  isolated  by 
itself. 

DUCLAUX'S  TORULA5 

This  yeast  was  isolated  from  milk  in  which  it  produced  an  active 
fermentation.  It  is  a  small,  ovoid  yeast.  Packets  which  may  reach 
large  size  are  formed  by  budding.  Inoculated  into  gelatin,  it  gives 
little  growth  and  this  is  almost  entirely  on  the  surface.  In  the  same 

1  Heinze,  B.,  and  Cohn,  E.     Ueber  Milchzuker  vergarende  Sprosspilze.     Zeit. 
Hygiene.     46,  1908. 

2  Beijerinck,  M.  W.     Sur  le  Kephir.     Arch,  neerlandaises  des  Sc.  exactes  et 
nat.,  23,  1889. 

3  Beijerinck,  M.  W.    Die  lactase  ein  neues  Enzyme.    Cent.  Bakt.  6,  1889. 

4  Freudenreich,  E.     Bakterien  Untersuchungen  iiber  den  Kefir.     Cent.  Bakt. 
3,  1897. 

6  DuClaux,  E.    Fermentation  ale.  du  sucre  de  lait.     Ann.  Past.  Inst.  1,  1887. 


308    NON-SACCHAROMYCETES   OR  DOUBTFUL  YEASTS 

medium  with  1  per  cent  of  glycerol  added,  it  produces  a  button- 
shaped  growth  and  a  little  development  along  the  line  of  inoculation. 
It  is  separated  from  the  yeast  of  Adametz,  which  will  be  mentioned 
later,  by  the  fact  that  its  branching  structure  is  less  developed.  In 
beer  wort,  similar  development  is  secured.  The  cells  elongate  and 
become  cylinders  (7ju  long).  The  yeast  grows  easily  in  milk  and  neu- 
tral serum  and  develops  especially  in  liquids  exposed  to  the  air  and 
with  difficulty  in  the  bottom  of  flasks.  The  aerobic  life  does  not 
hinder  its  enzyme  activity.  This  species  secretes  neither  rennin  nor 
casease  but  does  produce  lactase.  The  optimum  temperature  for 
fermentation  is  between  ,25  and  30°  C.  The  thermal  death  point  is 
50°  C.  for  cells  in  the  dry  state  and  60°  C.  for  moist  cells.  No  coagu- 
lation is  secured  in  milk.  An  alcoholic  drink  slightly  acid  and  with 
an  agreeable  taste  is  formed  from  milk.  It  ferments  easily  lactose, 
dextrose,  d-galactose,  saccharose  and  more  difficultly  maltose. 


TORULA  LACTIS.     (Adametz)  Heinze  and  Cohn 

This  species  was  isolated  from  a  spontaneous  fermentation  in  milk 
by  Adametz.1  It  has  oval  or  elliptical  cells  (7-10  JJL  X  5),  sometimes 
spherical  (3-4  jit),  larger  than  those  of  the  preceding  species.  The  buds 
are  usually  formed  simultaneously  at  both  of  the  opposite  ends  of  the 
ellipse.  On  the  plaster  block,  this  yeast  forms  no  ascospores  but 
the  cells  continue  to  bud  and  elongate.  On  peptone  gelatin  it  does 
not  do  well  and  gives  almost  entirely  a  superficial  growth.  The  colonies 
are  round  with  slightly  branching  edges  and  of  a  brown  color.  On 
peptone  gelatin  with  one  per  cent  of  glycerol  added,  in  stab  cultures, 
this  yeast  forms,  like  the  preceding,  a  superficial  button  along  the 
inoculation  growth  from  which,  in  fifteen  days,  fine  branches  extend. 
On  beer  wort  gelatin,  an  abundant  surface  growth  is  secured  in  which 
the  center  is  slightly  raised.  In  sterile  milk  fermentation  phenomena 
are  presented  in  24  hours  at  40°  C.,  in  48  hours  at  38°  C.,  and  in  4  days 
at  25°  C.  No  rennin  or  casease  is  formed.  In  beer  wort  it  grows  as 
a  sediment  at  the  bottom  of  the  flask  and  induces  a  manifest  fermen- 
tation. It  starts  a  faint  fermentation  in  milk  after  24  hours  a  little 
less  vigorous  than  that  caused  by  the  Torula  of  Duclaux.  It  fer- 
ments maltose  with  difficulty  but  ferments  easily  lactose,  d-galactose, 
dextrose  and  saccharose  (as  quickly  lactose  as  saccharose).  The  thermal 
death  point  of  dry  cells  is  between  50  and  60°  C.  and  of  moist  cells 
56°  C. 

1  Adametz,  L.  Saccharomyces  lactis,  eine  neue  Milchzucher  vergarende 
Hefenart.  Cent.  Bakt.  5,  1889. 


TORULA  COMMUNIS  309 

KAYSER'S  YEAST 

Secured  from  milk  at  a  farm  in  Brie,  Kayser  l  found  that  this  yeast 
possessed  cells  from  6  to  SJJL  long  and  3  to  5ju  wide.  It  forms  neither 
casease  nor  rennin.  Like  the  two  preceding  species,  it  ferments  sac- 
charose and  lactose  (the  lactose  as  easily  as  the  saccharose),  d-galac- 
tose,  and  dextrose  but  acts  with  difficulty  on  maltose.  The  thermal 
death  point  is  55°  C.  for  moist  cells  and  90-100°  C.  for  dry  cells.  On 
gelatin,  it  looks  like  the  preceding  yeast  and  is  distinguished  only  by 
the  fact  that  the  fibrous  appearance  is  less  pronounced. 

TORULA  COMMUNIS.     Browne 

Browne  2  has  found  a  Torula  the  most  abundant  organism  in  raw 
sugar  from  Cuba.  A  similar  organism  was  also  found  in  raw  sugar 
and  soft  refined  sugars  from  the  British  West  Indies.  Owen3  has  also 
mentioned  a  similar  organism.  The  colonies  on  raw  sugar  agar, 


Fig.  141-B.  —  Magnified  Cells  of  Torula  communis  (after  Browne). 

according  to  Browne,  appear  first  as  small  white  cysts  which  are  pointed 
under  the  microscope.  These  cysts  increase  in  size  to  a  diameter  of 
0.2-0.5  mm.  until  they  reach  the  surface  of  the  agar  after  which  they 
spread  out  in  all  directions.  The  colony  gradually  assumes  a  cir- 
cular shape  from  3-10  mm.  in  diameter  and  is  grayish  white  in  color. 
Old  colonies  are  brownish  in  color.  Under  high  power  of  the  micro- 
scope, no  mycelium  is  seen.  The  cells  are  separate  and  look  like 
yeasts.  Torula  communis  grows  readily  in  all  concentrations  of  sugar 
solutions.  A  granular  deposit  is  formed  and,  after  a  time,  a  thin 
marginal  scum.  There  seems  to  be  slight  evolution  of  gas.  No 
froth  or  foam  is  formed  as  is  often  present  with  strongly  fermenting 
organisms.  The  action  on  raw  sugar  seems  to  be  a  destruction  of  the 

1  Kayser,  E.     Contribution  physiologique  des  levures  alcooliques  de  la  lac- 
tose.   Ann.  Past.  Inst.  5,  1891. 

2  Browne,   C.   A.     The  Deterioration  of  Raw  Cane  Sugar.     A  Problem  in 
Food  Conservation.    J.  Ind.  Eng.  Chem.  10  (1918),  178-190. 

3  Owen.     Louisiana  Planter,  56,  173. 


310    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

invert  sugar,  fructose  being  the  constituent  most  strongly  attacked. 
Sucrose  is  not  inverted.  Data  are  produced  by  the  author  to  show 
that  this  organism  is  the  strongest  fermenter  between  the  ninth  and 
fifteenth  day.  Further  characteristics  of  this  organism  are  not  given 
by  Browne. 

LACTOMYCES  INFLANS  CASEIGANA.    Bochicchio 

Bochicchio  l  isolated  this  species  from  Lombard  cheese.  It  is  a 
top  yeast  with  unilateral  budding.  The  cells  are  elongated,  round  or 
elliptical.  When  cultivated  on '  gelatin,  this  yeast  forms  a  whitish 
colony  with  an  entire  edge.  It  coagulates  sterile  milk  and  liquefies 
part  of  the  coagulum  without  the  formation  of  distinct  amounts  of 
acid.  In  lactose  bouillon,  between  25  and  40°  C.,  it  provokes  an 
energetic  fermentation.  The  most  favorable  temperature  for  this  is 
situated  at  about  30°  C.  The  temperature  limit  is  about  60°  C.  Milk 
infected  with  this  yeast  is  changed  into  a  frothy  mass  with  a  dis- 
agreeable odor. 

SACCHAROMYCES  LEBENIS.     Rist  and  Khoury 

This  yeast  was  isolated  by  Rist  and  Khoury2  from  leben  where 
it  is  found  with  Mycoderma  lebenis.  It  possesses  oval  cells  (3  to  6ju) 
with  granular  contents.  The  cells  are  isolated  and  one  never  finds 
mycelial  formations.  On  sucrose  agar  below  the  surface,  this  yeast 
gives  little  growth  and  seems  to  grow  only  on  the  surface.  However, 
one  may,  by  successive  culturings  in  this  medium,  cause  it  to  take  on 
anaerobic  characteristics  and  the  fermentation  of  sucrose.  It  grows 
well  on  ordinary  gelatin  without  sugar  and,  at  the  end  of  48  hours, 
forms  white  circular  colonies  with  a  moist  and  damp  surface.  Stabs 
into  saccharose  gelatin  give  colonies  which  are  round  and  squeezed 
together  not  exceeding  3  to  4/z.  It  does  not  liquefy  the  gelatin.  On 
milk  broth  this  yeast  produces  a  cloudiness  and  later  a  sediment.  It 
causes  no  fermentation.  On  grape  must,  it  produces  a  cloudiness 
which  is  also  followed  by  a  deposit  in  the  bottom  of  the  culture  flask. 
This  species  ferments  saccharose  and  maltose  but  does  not  act  on 
lactose.  It  works  with  Mycoderma  lebenis  in  the  alcoholic  fermenta- 
tion of  lactose  but  it  acts  on  this  sugar  only  when  associated  with 
Streptococcus  lebenis  which  probably  decomposes  the  lactose. 

1  Bochicchio,   N.     Ueber  innen  Milchzucker  vergarenden  und  Kaseblahungen 
hervorrufenden  neuen  Hefenpilz.    Cent.  Bakt.  15,  1894. 

2  Rist,  E.,  and  Khoury,  J.    Etudes  sur  un  lait  fermente  comestible,  le  "leben" 
d'Egypte.    Ann.  Past.  Inst.  16,  1902. 


DOMBROWSKFS  TORULA  311 


TORULA  KEPHIR.     Nokolajewa l 

This  species  was  found  along  with  many  bacteria  and  Torula 
ellipsoidea  in  a  decomposition  of  kephir.  It  is  made  up  of  round 
cells  (3-4 n  in  diameter);  it  develops  with  a  red  color  on  potato. 
This  yeast  ferments  dextrose,  saccharose  and  lactose. 


TORULA  ELLIPSOIDEA.     Nikolajewa2 

This  yeast  was  found  under  the  same  conditions  as  the  preceding. 
The  cells  are  elliptical  (6-9 /i  in  length  and  3-4. 5 /z  in  width)  and  de- 
velop on  all  substances.  A  yellow  pigment  is  formed  on  potato. 
This  yeast  ferments  dextrose  and  saccharose  but  not  lactose 


TORULA  AMARA.     Harrisson3 

This  yeast  was  isolated  from  a  cheese  and  milk  in  America  where 
it  produced  a  bitter  taste.  Harrisson,  who  isolated  it,  showed  that  it 
came  from  cans  of  milk;  the  cans  became  infected  from  trees  under 
which  they  were  placed  to  dry.  It  produces  a  bad,  disagreeable  taste 
in  milk  at  37°  C.  after  14  hours.  It  produces  an  odor  recalling  that  of 
plum  stones.  The  taste  is  astringent.  Later  the  milk  coagulates  and 
an  aromatic  ethereal  odor  is  formed.  The  optimum  temperature  for 
budding  is  37°  C.  and  the  temperature  limits  48-50°  C.  This  species 
easily  ferments  saccharose,  dextrose  and  lactose.  It  grows  in  a  bouil- 
lon containing  2.4  per  cent  of  lactic  acid. 


DOMBROWSKTS  TORULA 

Torula  lactis  a,  Dombrowski:  This  yeast  was  isolated  from 
Armenian  mazun  in  Zurich  by  Dtiggeli  and  was  described  by  Dom- 
browski.4 The  cells  are  usually  oval;  giant  cells  are  often  noticed  in 
hanging  drop  preparations.  On  gelatin  plates,  the  colonies  are  lentic- 
ular and  are  either  circular  or  torpedo  shaped.  Growth  in  gelatin 
stabs  extends  only  4  cm.  below  the  surface.  The  giant  colony  is  flat 
and  spread  out  with  a  slightly  fringed  border.  In  beer  wort  and  must, 
this  species  acts  like  a  top  yeast.  Fermentation  is  quite  energetic. 

1  Nikolajewa,  E.     Die  Microorganismen  des  Kefirs.     Bull.  Jard.  Imp.  St. 
Petersburg,  7,  1907. 

2  See  reference  for  Torula  kephir. 

3  Harrisson,  F.  C.    Bitter  milk  and  cheese.    Cent.  Bakt.  9,  1902. 

4  Dombrowshi,  W.     Sur  TEndomyces  fibuliger.     Comp.  Rend.  d.  trav.  du. 
labor,  de  Carlsberg.  7,  Book  4,  1909, 


312    NON-SACCHAROMYCETES   OR  DOUBTFUL  YEASTS 

The  must  is  strongly  discolored  with  the  formation  of  an  aroma.  A 
scum  is  not  formed,  only  a  feeble  ring.  Clarification  is  generally  bad. 
At  the  end  of  five  and  a  half  months  in  wort  about  5  per  cent  of  alco- 
hol is  formed.  This  yeast  induces  an  active  fermentation  in  wort 
with  the  formation  of  an  aroma.  It  ferments  dextrose,  lactose,  sac- 
charose and  d-galactose  but  has  no  action  on  maltose.  Besides  alco- 
hol and  carbon  dioxide,  it  produces  a  small  quantity  of  acid. 

Torula  lactis  ft  Dombrowski:  This  species  was  isolated  by  Burri 
and  described  by  Dombrowski.  It  possesses  cells  of  varied  shapes. 
In  solid  media,  they  are  generally  elongated  and  united  by  a  sort  of 
mycelium.  In  wort  cultures,  they  are  spherical,  elliptical,  elongated 
or  oval.  The  average  dimensions  are  7-9. 5 /z  in  length  and  4.25- 
4.5  jit  in  width.  Giant  cells  are  formed  in  hanging  drops. 

The  colonies  on  gelatin  or  beer  wort,  in  plates,  are  either  torpedo 
shape  or  circular.  They  are  made  up  of  elongated  cells  resembling  a 
mycelium.  In  stabs,  development  extends  to  about  4  cm.  below  the 
surface.  The  giant  colony  has  a  concavity  in  the  center.  Cultures 
on  beer  wort  show  a  ring  formation  and  a  feeble  attempt  to  form  a 
scum.  The  wort  is  strongly  discolored.  There  is  the  production  of  a 
slight  aroma.  After  five  and  a  half  months,  there  are  6.3  grams  of  alco- 
hol formed  in  100  c.c.  of  medium.  This  species  produces  at  25°  C. 
an  active  fermentation  of  milk  with  a  slight  disagreeable  taste.  It 
ferments  lactose,  saccharose,  d-galactose  and  dextrose  but  does  not 
act  on  maltose.  Small  quantities  of  acid  are  produced  in  the  fermen- 
tation. 

Torula  lactis  7,  Dombrowski:  This  species  was  found  many  times 
in  kephir  grains.  It  possesses  oval  cells,  sometimes  spherical,  which 
have  a  diameter  on  beer  wort  of  3.5 /z,  and  which  often  possess  numer- 
ous fat  globules.  Colonies  on  beer  wort  or  gelatin  plates  are  circular 
or  shaped  like  torpedoes.  In  stab  cultures  growth  extends  about  4.5 
cm.  below  the  surface.  Giant  colonies  possess  a  concavity  in  the  center. 
This  species  produces  a  rather  thick  scum  on  beer  wort  which  is  of  a 
whitish  color  and  forms  about  5  grams  of  alcohol  in  about  five  and  a 
half  months.  It  acts  like  a  top  yeast.  It  clarifies  beer  wort  and  pro- 
duces an  active  fermentation.  The  wort  is  strongly  discolorized  with 
the  formation  of  an  aroma.  At  23-25°  C.,  this  Torula  causes  an  active 
fermentation  in  milk.  It  ferments  lactose,  saccharose,  dextrose  and 
d-galactose  but  does  not  act  on  maltose.  Small  amounts  of  acids  are 
produced. 

Torula  lactis  d  and  Torula  lactis  e,  Dombrowski:  These  two 
species  were  encountered  in  various  products  from  milk.  They  are 
only  distinguished  by  the  size  of  the  cells.  The  cells  are  spherical 
in  shape  and  often  include  a  large  fat  globule.  The  cells  of  Torula 


ROGER'S  TORULA  313 

lactis  5  are  much  smaller  than  those  in  Torula  lactis  €.  In  the  first 
they  are  2.5-4. 12 /z  and  in  the  other  3. 1-5.6 /z.  On  gelatin  in  beer 
wort,  in  plates,  both  species  form  spherical  or  torpedo-shaped  colonies. 
The  giant  colonies  of  Torula  lactis  5  have  almost  a  flat  surface  while 
the  others  possess  a  concentrically  folded  surface.  In  beer  wort 
or  grape  must,  both  species  produce  a  fine  ring  but  cause  no  fermenta- 
tion. The  wort  is  not  discolored  and  there  is  no  formation  of  an  aroma. 
This  does  not  ferment  milk.  Neither  does  it  ferment  lactose,  saccha- 
rose, dextrose,  d-galactose  and  maltose. 

Torula  No.  15,  Dombrowski:  The  shape  of  the  cells  is  oval.  On 
plaster  blocks,  the  cells  possess  a  large  fat  globule.  On  beer  wort 
gelatin  plates,  this  species  produces  circular  or  torpedo-shaped  colo- 
nies. The  giant  colonies  show  slight  development  with  concentric 
zones.  In  carbohydrate  liquid  media,  this  species  produces  no  fer- 
mentation but  develops  abundantly.  At  23-25°  C.  the  scum  is  a 
bright  red.  This  yeast  produces  a  strong  cloudiness  and  a  disagree- 
able odor.  Many  other  milk  yeasts  have  been  isolated  by  Pierroton 
and  Riboni  Weigmann,  Kalanthar,  Jensen,  and  Maze;  they  are  too 
insufficiently  known  to  be  described  here. 


C.     YEASTS  FROM  FATS 

SACCHAROMYCES   OLEI.    Van  Tieghem 1 

This  yeast  was  accidentally  observed  by  Van  Tieghem  in  olive  oil 
in  which  there  were  entrained  droplets  of  water.  It  possesses  oval 
cells  arranged  like  heads  in  a  chain.  These  bead-like  structures  break 
off  and  the  isolated  cells  bud  in  order  to  form  new  ones.  The 
cells  measure  on  an  average  4/z  and  2.5  JJL.  Their  contents  is  a  rose 
color.  This  yeast  develops  in  all  stretches  of  the  medium  without 
growing  on  the  surface.  The  oil  undergoes  a  marked  change,  becoming 
acid  and  saponifying. 

ROGER'S  TORULA 

This  yeast  was  isolated  from  different  samples  of  butter  by  Rogers.2 
It  possesses  the  property  of  decomposing  fats  with  the  formation  of 
fatty  acids.  The  cells  are  elliptical  (3  to  5/*)  and  show  a  slight 
tendency  to  form  chains  or  masses.  It  ferments  maltose  slowly  but 
does  not  act  on  other  sugars  (lactose,  d-mannose,  levulose,  dextrose). 

1  Van  Tieghem.      Sur  la  vegetation   dans  Fhuile.     Bull.   Soc.   Bot.   France. 
28,  1881. 

2  Rogers,  A.     Eine  gespaltende  Torulahefe  aus  Buchsenbutter  isoliert.     Cent. 
Bakt.  10,  1903. 


of 


314    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 


D.      COLORED  TORULA 

Among  the  colored  Torula,  the  red  yeasts  are  the'  most  numerous.1 
They  are  especially  numerous  in  dust  of  the  air.  Some  form  myco- 
dermic  scums  and  may  be  classed  as  Mycoderma. 


TORULA  PULCHERRIMA.     Lindner 


Lindner2  has  found  this  Torula 


Fig.  142.  —  Torula  pulcherrima.     Old  Cells 
and  Their  Germination  (after  Lindner). 

membrane  ruptures  itself  and  an 
yeast  ferments  dextrose,  d-mannose 


on  numerous  occasions,  especially 
on  various  fruits  and  also  on  the 
excrement  of  potato  bugs.  Red 
pigments  are  formed.3  In  beer 
wort,  its  cells  are  at  first  ellipti- 
cal but  later  they  become  larger 
and  round  with  a  large  fat  glob- 
ule in  their  interior.  They  possess 
a  thick  membrane  (Fig.  142). 
During  germination  the  external 
active  budding  takes  place.  This 
and  levulose. 


TORULA  MUCILAGINOSA.    Jorgensen 4 

The  cells  are  oval  (5  to  5.6  ju,  long  and  2/z  wide).  Inoculated  into 
beer  wort,  this  yeast  at  first  produces  a  slight  cloudiness  and  at  the 
same  time  a  ring  of  a  mucous  yeast  with  a  rose  color  as  well  as  a 
mucous  sediment  visible  only  after  shaking  the  culture.  The  ring 
continues  to  extend  on  the  walls  of  the  vessel  which  soon  finds  itself 
covered  from  top  to  bottom  with  a  rose-colored  growth.  There  seems 
to  be  no  vegetation  as  a  sediment.  Clumps  of  this  mucous  ring  may 
fall  to  the  bottom  of  the  flask.  The  surface  colonies  on  gelatin  with 

1  per  cent  wort  are  round,  faintly  rose  colored,  moist,  shiny  and  a 
little  convex.     The  young  colonies  have  a  united  border.     The  old 
ones  are  hollowed  in  the  middle  and  provided  with  little  transverse 

1  Beijerinck   has   described   a    Saccharomyces   pulcherrimus   which   secretes   a 
colorless  chromogen  which  becomes  a  deep  red  in  the  presence  of  iron  salts.     He 
even  suggests  that  this  variety  may  be  used  as  a  test  for  iron.     (Beijerinck,  M.  W. 
Chromogenic  yeasts  —  a  new  biologic  reaction  for  iron.     Arch,  neerland.  physiol. 

2  (1918),  609-15.     Chem.  Absts.  13  (1919),  1082. 

2  Lindner,   P.     Ueber  rot  und  Schwartz  gefarbte  Sprosspilze.     Wochenschr. 
f.  Brau.  4,  1887. 

3  All  of  the  red  yeasts  have  been  grouped  by  bacteriologists  into  a  special 
group  known  as  "red  yeasts." 

4  Jorgensen,  A.     Die  Mikroorganismen  der  Garungsindustrie.      Berlin.     5th 
edition.    Paul  Parey.    1909. 


RED  TORULA  315 

furrows  at  the  edges.  This  yeast  produces  no  fermentation  of  dex- 
trose, maltose,  lactose,  saccharose,  raffinose  and  dextrine.  It  inverts 
saccharose  and  decomposes  raffinose.  In  must  with  added  alcohol, 
it  forms,  at  the  end  of  8  days,  a  mucous  ring  if  the  alcohol  does  not 
exceed  2  per  cent.  With  5  per  cent  of  alcohol,  there  is  no  develop- 
ment. The  formation  of  a  mucous  ring  seems  to  be  related  to  the 
presence  of  albumin  in  the  medium  and  concerned  with  the  presence 
of  carbohydrates.  It  increases  when  the  amount  of  peptone  added 
to  the  medium  is  increased. 

TORULA  CINNABARINA.    Jorgensen l 

This  yeast,  improperly  designated  under  the  name  of  Torula, 
seems  to  belong  to  the  Mycoderma.  The  cells  are  oval  or  elongated 
often  provided  with  short  or  long  tubes,  a  sort  of  promycelium. 
Giant  cells  are  often  noticed  either  elon- 
gated or  round.  The  long  ones  may  be 
14.6/j  in  length  and  the  round  ones  9.5/z 
in  diameter.  Cultivated  in  must  or  in 
solutions  of  the  various  sugars,  this  yeast 
produces  a  scum  which,  at  first,  is  united,  \) 
folded  and  of  a  red  color.  The  liquid 
remains  clear.  No  sediment  is  noticed  at 
the  bottom  of  the  culture  flask  nor  any  Fig  143 
fermentation.  In  old  cultures,  the  wort  Scum  on  Old  Culture  (after 
undergoes  a  notable  decoloration.  At  the  oreensen)- 
end  of  60  hours  at  25°  C. ,  small  islands  of  floating  scum  are  produced 
in  which  a  small  number  of  cells  begin  to  form  a  mycelium. 

At  the  end  of  24  hours,  the  formation  of  a  promycelium  may 
become  very  abundant.  On  the  promycelium  and  on  the  mother 
cells,  the  formation  of  buds  may  be  seen.  (Fig.  143.)  The  surface 
colonies  on  gelatin  with  10  per  cent  of  wort  are  round,  with  a  faint 
red  color.  The  old  colonies  are  dry  and  show  concavity  and  a  finely 
fringed  border.  This  yeast  produces  no  fermentation  in  dextrose, 
maltose,  lactose,  saccharose,  raffinose  nor  dextrine.  It  decomposed 
solutions  of  saccharose  and  raffinose.  In  wort  with  1  to  2  per  cent 
of  alcohol  added,  there  is  a  feeble  development.  If  one  decreases 
these  amounts  of  alcohol,  the  yeast  ceases  to  grow. 

RED   TORULA,  NO.   36.    Janssens  and  Mertens 

This  is  a  yeast  a  little  smaller  than  S.  pastorianus  which  in  its 
scums  seems  to  have  a  tendency  to  form  elongated  cells  and  filaments. 

1  Jorgensen,  A.    See  reference  for  Torula  mucilaginosa. 


316    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

It  was  found  in  the  bottom  of  a  bottle  of  Maidstone  beer  and  de- 
scribed by  Janssens  and  Mertens.1  On  beer  wort  it  develops  on  the 
surface  and,  after  two  days,  produces  a  red  scum.  This  develops  very 
quickly  and  covers  the  whole  surface  of  the  liquid,  later  to  become 
thick  and  folded.  It  also  forms  a  reddish  ring  on  the  walls  of  the 
culture  flask.  The  scum,  if  one  shakes  the  culture  flask,  falls  to  the 
bottom  and  is  replaced  by  a  new  one.  If  ammonium  carbonate  to  2 
per  cent  is  added  there  is  no  formation  of  this  scum.  The  cells  go  to 
the  bottom  of  the  liquid  and  the  solution  becomes  cloudy.  Only 
when  all  of  this  ammonium  carbonate  has  been  destroyed  does  the 
scum  form  again. 

On  gelatin  plates,  this  yeast  produces  surface  colonies  at  the 
end  of  two  days,  visible  to  the  naked  eye.  After  5  days,  these  colonies 
are  entirely  developed  and  possess  a  very  characteristic  appearance. 
There  is  a  little  enlargement  in  the  middle  and  there  is  formed  along 
their  peripheries  a  slight  fringe.  .  This  Torula  liquefies  gelatin  very 
slowly.  The  optimum  temperature  for  budding  is  situated  between 
20  and  25°  C.  Toward  30°  C.,  the  vitality  of  the  yeast  is  somewhat 
diminished.  This  species  produces  no  alcoholic  fermentation  and  is 
not  pathogenic. 

The  red  pigment  is  almost  insoluble  in  water  and  acetone  but  is 
quite  soluble  in  carbon  bisulfide.  It  seems  to  resemble  carotine. 

TORULA  GLUTINIS.     Pringsheim  and  Bilewsky 

Syn.:  CRYPTOCOCCUS  GLUTINIS.    Fresenius.    SACCHAROMYCES 
GLUTINIS.      Cohn 

Fresenius 2  discovered  this  yeast  which  is  very  common  in  dust 
of  the  air.  It  is  a  common  red  yeast  and  has  been  since  encountered 

by  Cohn  and  Schroter.3    Hansen  4  has 

%    c®@     O   *  s\  r\      also  studied    it    under  the    name   of 

§  •£)    @     w    v    @  £T     Cryptococcus  glutinis.     He  found  that 

Fig.  144.— S.glutinis  (after  Hansen).   many    species    have    been   described 

under    this    name    and    that    Cohn's 

yeast  does  not  correspond  with  that  described  by  Frecenius. .  Hansen 
has  isolated  many  other  species  of  red  yeasts  related  to  the  C.  glutinis 

1  Janssens,  E.   A.,   and    Mertens,   A.     fitude  microchemique   et  cytologique 
d'une  Torula  rose.    La  cellule,  20,  1903. 

2  Fresenius.    Beitrage  zur  Mycologie.     1850. 

3  Cohn,   F.,    and  Schroter,   J.     Beitrage  zur  Biologic  der  Pflanzen.     Vol.    1. 
1872. 

4  Hansen,  E.  C.    Saccharomyces  colores  en  rouge  et  cellules  rouge  ressemblant 
a  de  Saccharomyces.     Comp.  Rend,  des  trav.  lab.  de  Carlsberg.     Vol.  1,  Book, 
24,  1879. 


TORULA  GLUTINIS  317 

of  Fresenius.  One  seems  to  correspond  to  the  species  described  by 
Cohn  and  the  other  to  a  true  Saccharomyces  possessing  ascospores 
and  a  third  presents  in  beer  wort  budding  cells  like  a  true  yeast  but 
also  develops  a  promycelium  or  germinating  tube  when  it  exists  in  a 
state  of  poor  nutrition.  (Fig.  145.) 

Sartory !   in    1907  reported  a  red  ,  g       o     0     A    R   /?    «v 

yeast  which  he  compared  to  the  «X  -Q^  J(  \^  "^  "0  ^J  "0 
species  of  Fresenius.  It  is  a  yeast 
very  widespread  in  nature  and 
which  one  may  find  in  macerations 
of  grains,  the  rinds  of  certain 
cheeses  and  other  organic  sub- 
stances. The  cells  are  oval,  the 
average  dimension  being  5  to  llpt 
X  4  jtt.  The  optimum  temperature 

for  budding  is  between  22°  C.  and 

one  n        A  j.   OT  4.     ooo  r\      ,i  ,    Fig.   145.  —  Rose  Yeast  Related  to  S. 

30   C.     At  37  to  38    C.,  the  yeast  glutinis  (after  Hansen). 

stops  vegetating. 

On  glycerol  broth,  it  forms  a  scum  made  up  of  cells  which  are 
associated  to  form  a  sort  of  mycelium.  The  sediment  is  made  up  of 
oval  cells.  On  carrot,  this  yeast  develops  rapidly,  giving  a  red  layer. 
On  plain  potato,  acid  or  glycerol,  and  on  artichoke  the  develop- 
ment is  less  rapid.  Small  colonies  are  formed  which  have  a  reddish 
color.  On  gelatin  and  agar,  the  vegetation  is  less  abundant  and  there 
is  produced,  after  a  certain  time,  a  liquefaction  of  the  gelatin.  This 
yeast  secretes  invertase  but  produces  no  alcoholic  fermentation.  It 
is  without  action  on  maltose,  d-galactose,  starch  and  inuline.  On 
milk,  in  about  14  days,  there  is  a  precipitation  of  the  casein  with  no 
peptonization. 

Quite  recently,  Pringsheim  and  Bilewsky  have  isolated  another 
red  yeast  which  is  much  like  Cryptococcus  glutinis  of  Fresenius  which 
was  named  Torula  glutinis.  This  yeast  has  no  agreement  with  the 
yeast  of  Sartory. 

The  cells  are  spherical  or  oval  (5  to  6/x  in  length  and  4  to  5  ;u  in 
width),  isolated  or  united  in  budding  formation  but  easily  separable. 
They  possess  small  granules  and  one  or  two  large  globules  of  fat.  In 
culture,  the  cells  possess  a  reddish  color  which  may  become  brown 
under  unfavorable  conditions.  The  optimum  temperature  for  budding 
is  between  6°  and  15°  C.  The  minimum  is  about  0  and  the  maxi- 
mum near  47°  C.  The  cells,  in  the  vicinity  of  the  minimum  and  maxi- 
mum temperature,  are  very  small.  Under  certain  conditions,  giant 

1  Sartory,  A.  fitude  biologique  du  Cryptococcus  glutinis.  Bull,  de  la  myc. 
de  France,  23,  1907. 


318    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

cells  with  a  diameter  of  10-25  ju  are  formed  along  with  long  budding 
cells,  incompletely  formed  and  irregular.  In  liquid  media  the  yeast 
forms  a  thin  pellicle  on  the  surface  and  a  deposit  in  the  bottom  of 
the  flask.  On  solid  substrates,  it  forms  small  dots  of  growth  about 
0.5  to  1/i  in  diameter.  Later  these  run  together  forming  a  shiny 
mass  almost  mucous.  The  giant  colonies  on  potato  present  a  wrinkled 
appearance.  On  agar  and  gelatin,  in  streaks  and  stabs,  the  vegetation 
is  at  first  with  an  even  edge  which  after  a  certain  time  becomes 
furrowed.  Torula  glutinis  does  not  possess  a  very  characteristic  ap- 
pearance and  a  series  of  related  yeasts  have  been  described  under 
this  name. 

CRYPTOCOCCUS  BAINIERI.    Sartory 

This  yeast  was  found  by  Bainier  on  the  stems  and  leaves  of  the 
nettle  where  it  lived  as  a  saprophyte.  It  has  been  described  by  Sar- 
tory.1 It  is  easily  cultivated  on  all  solid  media  (gelatin,  agar,  potato, 
both  acid  and  glycerol),  and  especially  on  carrot.  The  colonies  are  of 
a  beautiful  deep  rose  color.  On  certain  sugar  media  the  color  becomes 
a  poppy  colored  red.  The  yeast  gives  abundant  growth  in  liquid 
media  (RauhVs  solution,  maltose,  lactose,  galactose  or  glycerine, 
Raulin's  solution)  and  especially  on  glycerol  bouillon.  The  optimum 
temperature  for  budding  is  situated  between  24  and  26°  C.  The 
development  begins  at  15°  C.  and  stops  at  38°  C.  to  40°  C.  This  yeast 
produces  between  15°  and  36°  C.  a  rose-colored  scum  made  up  of  elon- 
gated cells  of  larger  dimensions  than  the  cells  in  the  sediment.  It 
secretes  invertase  but  does  not  ferment  dextrose,  maltose,  lactose 
nor  d-galactose. 

PSEUDOSACCHAROMYCES   STEVENSI.    Anderson2 

Anderson  isolated  this  yeast  from  human  feces  and  characterized 
it  as  follows: 

"  Morphology.  In  both  young  and  old  cultures  the  cells  are  narrowly 
elliptical,  oblong  or  apiculate;  cytoplasm,  very  granular;  vacuoles, 
not  distinct  except  in  old,  swollen  cells;  no  elongated  cells  or  false 
mycelium  are  formed  under  any  condition  of  culture.  Budding  occurs 
only  at  ends,  by  elongation  and  swelling  of  the  apiculate  portion. 
The  size  is  2  X  5  jit.  No  endospores  are  formed. 

"  Cultural  Characters.     On  glucose  agar  the  streak  is  filiform,  glisten- 

1  Sartory.     fitude  d'une  levure  nouvelle,  le  Cryptococcus  Bainieri.     Comp. 
Rend.  Soc.  de  Biol.  61,  1906. 

2  Anderson,  H.  W.     Yeast-like  fungi  of  the  human    intestinal  tract.     Jour. 
Inf.  Diseases,  21  (1917),  341-386. 


CRYPTOCOCCUS  VERRUCOSUS  319 

ing,  white,  flat,  and  smooth.  The  growth  is  slow,  and  the  colony 
becomes  dirty-gray  with  age.  In  gelatin  no  liquefaction  occurs; 
the  growth  is  filiform.  In  beer  wort  and  sugar  mediums  there  is  slow 
development,  with  no  evidence  of  growth  except  a  slight  sediment.  The 
giant  colonies  are  very  small. 

"Physiologic  Properties.  There  is  no  fermentation  of  glucose, 
levulose,  sucrose,  lactose,  raffinose,  galactose  or  maltose.  No  decided 
change  in  acidity  occurs  in  these  sugars,  dextrin  or  yeast  water.  There 
is  no  change  in  litmus  milk. 

CRYPTOCOCCUS  VERRUCOSUS.    Anderson1 

"Morphology.  In  young  liquid  culture  the  cells  are  oblong, 
narrowly  elliptical  or  oblong-elongated;  in  old  cultures  elongated  cells 
are  common,  with  several  'oil '  globules  in  each  cell.  The  size  is 
3X9  microns.  Budding  occurs  from  shoulders,  ends  or  sides.  No 
endospores  are  formed. 

"Cultural  Characters.  On  glucose  agar  slant  there  is  at  first  an 
even,  filiform,  glistening,  white,  smooth  growth;  later  it  becomes  dull, 
brittle,  verrucose  and  pulvinate. 
On  carrot  slant  the  growth  is  more 
profuse,  with  verrucose,  and  pul- 
vinate. On  carrot  slant  the  growth 
is  more  profuse,  with  verrucose 
character  more  pronouned,  and 
with  chalky-white  surface.  There 

is  a  filiform  or   nodose  growth  in  ~      1/IK  A       r 

.     .  .  .  T        r       •          FlS-     145-A.  —  Cryptococcus   verrucosus, 

gelatin  stab,  with  no  liquefaction.  Anderson. 

On    SUgar  mediums  and    beer    WOrt,     1,  Cells  from  Young  Beer  Wort  Culture;   2,  Old 

after  2  days,   a  few   small,  white 

patches  appear  on  the  surface,  later  becoming  larger,  dry  and  very 

firm;   at  first  they  remain  separate,  but  later  coalesce. 

"Physiologic  Properties.  It  does  not  ferment  glucose,  levulose, 
sucrose,  maltose,  galactose,  lactose  or  raffinose.  No  decided  change 
in  acidity  occurs  in  these  sugars.  Litmus  milk  becomes  very  slightly 
alkaline  after  several  weeks. 

"The  culture  was  isolated  from  human  feces. 

"The  dry  brittle  character  of  the  colonies  on  solid  mediums,  the 
formation  of  the  isolated,  white  patches  on  all  liquid  mediums,  and 
the  peculiar  type  of  cells,  clearly  distinguishes  this  yeast  from  any 
other  studied." 

1  Anderson,  H.  W.     Yeast-like  fungi   of  the  human  intestinal  tract.     Jour. 
Infectious  Diseases,  21  (1917),  341-386. 


320    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

CRYPTOCOCCUS   OVOIDEUS.     Anderson 

"Morphology.  Cells  in  young  cultures  are  round  or  oval,  and 
fairly  uniform  in  size  and  shape;  in  old  culture  cells  are  oval  or 
broadly  elliptical,  varying  markedly  in  size  and  with  few  budding  cells. 
There  are  no  elongated  cells  or  hyphal  elements.  The  size  is  3.5  X  4.5/z. 
"Cultural  Characters.  On  glucose  agar  the  streak  is  filiform, 
slightly  raised,  glistening,  smooth,  and  chalk-white.  The  growth  is 

slow  and  there  is  little  change  in 
cultures.     There  is  a  filiform 
^UY^r\     growth    in    gelatin  stab,  with  no 
(-&Q  0       liquefaction.     No  pellicle  or  ring  is 
O  present  in  beer  wort  or  in  liquid 

2  sugar  mediums. 

Fig.     145-B.- -Crptocomis     ovoideus,          "Physiologic  Characters.     There 


8 


1,  Cells  from  Young  Beer  Wort  Culture;   2,  Cells     IS    slight    fermentation     of     gluCOSC, 

levulose,  and  sucrose.  This  occurs 

only  after  a  week  and  the  production  of  gas  is  never  over  10  per  cent 
of  the  closed  arm  of  the  tube.  No  decided  change  in  acidity  occurs 
in  sugar  mediums.  There  is  no  change  in  litmus  milk. 

"The  culture  was  isolated  from  human  feces. 

"This  species  is  very  similar  in  many  of  its  characters  to  Culture 
170.101.  The  latter,  however,  ferments  glucose  and  levulose  very 
rapidly  and  completely.  Both  of  these  cultures  are  slow  growing, 
very  smooth  and  remain  white  and  even-edged  in  very  old  cultures. 
The  surface  elevation  is  not  so  decidedly  convex  as  in  most  yeasts  of 
the  white,  glistening  type." 

CRYPTOCOCCUS  GLABRATUS.     Anderson1 

"  Morphology.  Cells  in  young  cultures  are  oval  or 
elliptical,  and  fairly  uniform  in  size  and  shape;  in  old 
cultures  cells  are  round,  oval,  or'  elliptical  and  more 
variable  in  form  and  size.  Budding  occurs  from  the  ends 
or  shoulders  of  the  oval  and  elliptical  cells.  There 
are  no  elongated  cells  or  hyphal  elements.  The  size  is 
3  X  4.5  /A. 

"Cultural  Characters.  On  glucose  agar  the  streak  is 
filiform,  glistening,  raised,  smooth,  and  chalk-white.  In 
old  cultures  the  surface  remains  smooth  and  the  edge 
entire.  There  is  a  slow  growth  on  all  solid  mediums;  i,  ceils  from  Young 
liquid  mediums  remain  clear  with  little  evidence  of 
growth,  and  no  pellicle  or  ring  formation  is  present. 

1  Anderson,  H.  W.    See  reference  for  Cryptococcus  verrucosus. 


KRAMER'S   RED   TORULA  321 

"Physiologic  Characters.  There  is  rapid  fermentation  of  glucose 
and  levulose.  Other  sugars  are  not  fermented.  Litmus  milk  becomes 
only  slightly  alkaline.  No  decided  change  in  acid  reaction  occurs  in 
sugar  mediums.  Gelatin  is  not  liquefied. 

"The  culture  was  isolated  from  human  feces. 

"This  species  differs  in  few  respects  from  Cryptococcus  ovoideus. 
The  cells  are  more  elliptical  and  the  fermentation  reactions  are  unlike. 

CRYPTOCOCCUS  AGREGATUS.     Anderson1 

"Morphology.  In  both  young  and  old  cultures  the  cells  are  mostly 
globular  or  slightly  oval.  No  elongated  cells  are  formed.  Budding 
occurs  from  any  point  on  the  cell;  usually  several  buds  arise  from 
each  cell ;  in  old  cultures  buds  are  commonly  formed  in  large  numbers 
about  a  single  enlarged  cell.  The  size  is  3.5ju. 

"Cultural  Characters.  On  glucose  agar  slant  the  growth  is  filiform, 
convex,  glistening,  smooth,  chalk-white  and  firm.  In  old  cultures 
the  surface  remains  smooth, 

with    even    edges    and    no       ^     rQ'rC)  oU 

darkening    in    color.     Fili-       ^       xT-O  0°/Q 

form,  later  nodose,   growth      rOn  r  I/V  OO   o  O 


occurs  in  gelatin  stab,  with 


no  liquefaction.    No  pellicle 
or  ring  is  formed  in  beer  wort 

or    liquid    sugar    mediums. 

3  Fig.  145-D.  —  Crytococcus  agregatus,  Anderson. 

The      Surface      Of     the      giant         i,  Cells  from  Young  Beer  Wort  Culture;  2,  Cells  from 

colonies    on    glucose     agar 

plates  remains  remarkably  smooth,  only  dim,  concentric  lines  appearing. 

"Physiologic  Properties.  There  is  no  fermentation  in  glucose, 
sucrose,  levulose,  maltose,  galactose,  lactose  or  raffinose  yeast  water. 
No  decided  change  in  acidity  occurs  in  these  sugar  mediums.  Litmus 
milk  becomes  very  slightly  alkaline  after  3  weeks. 

"The  culture  was  isolated  from  human  feces. 

"Two  other  cultures,  isolated  from  the  same  person,  were  compared 
with  the  foregoing  species  and  found  to  be  identical.  The  isolations 
were  made  from  the  same  sample  of  feces  but  from  different  colonies." 

KRAMER'S   RED   TORULA 

This  species  found  by  Kramer2  in  cider  is  a  yeast  which  produces 
a  top  fermentation.    It  is  provided  with  a  red  pigment  soluble  in  water. 

1  Anderson,  H.  W.     See  reference  for  Cryptococcus  verrucosus. 

2  Kramer,    E.      Ueber   einen   rotgefarbten   bei   Vergarung    des    Mostes    Mit- 
wirkenden  Sprosspilz.    Osterr.  landw.    Cent.  1,  1891. 


322    NON-SACCHARQMYCETES   OR  DOUBTFUL  YEASTS 

It  ferments  dextrose  and  in  a  10  per  cent  solution  of  this  sugar  pro- 
duces 4.5  per  cent  of  alcohol  by  volume.  It  inverts  saccharose  and 
ferments  maltose.  It  has  no  action  on  lactose. 


SACCHAROMYCES   JAPONICUS.     Yabe1 

This  yeast  was  isolated  from  some  swampy  fields  in  Japan  on  rice 
leaves.  It  is  frequently  encountered  as  is  S.  keiskenna  in  dust  of  the 
air  in  Japan.  The  cells  are  elliptical  and  slightly  rounded.  In  Pas- 
teur's medium,  they  measure  6  X  3^t;  in  meat  bouillon,  9.2  X  5/y, 
sometimes  reaching  10.3  X  6.1 /x.  The  budding  is  accomplished  by  a 
special  method.  The  cells  send  out  a  long  tubule  about  twice  as 
long  as  the  cell  at  the  end  of  which  there  develops  an  enlargement 
constituting  the  bud.  In  certain  cases,  especially  in  peptone  broth, 
this  filament  branches  in  place  of  budding  and  gives  mycelial  forma- 
tions. This  produces  a  red  scum  on  liquids  which  falls  to  the  bottom 
of  the  flask  when  disturbed.  Stab  cultures  on  carbohydrate  gelatin 
after  a  few  weeks  show  along  the  line  of  inoculation  a  feeble  trace 
of  growth.  On  the  surface,  on  the  contrary,  a  reddish  pellicle  is  formed 
which  develops  progressively  and  liquefies  the  gelatin.  This  yeast  is 
essentially  aerobic  producing  no  fermentation.  The  red  pigment  ap- 
pears only  in  contact  with  air  and  is  especially  formed  in  cultures  on 
potato.  Saccharose  and  dextrose  are  good  foods  for  this  yeast,  better 
than  lactose.  Alcohol  to  3  per  cent  retards  development;  7  per  cent 
of  alcohol  prevents  it.  The  cells  die  in  5  minutes  at  45°  C. 


SACCHAROMYCES  KEISKEANA.     Yabe 

This  yeast  was  found  by  Yabe  2  along  with  the  preceding  one.  Its 
cells  are  of  a  pale  reddish  color  and  are  always  spherical  (5.1/Lt  in 
diameter).  Under  good  conditions  of  nutrition  they  may  reach  9/z. 
The  cells  grow  by  a  budding  analogous  to  that  of  bottom  beer  yeasts. 
No  mycelial  formation  exists.  In  stab  cultures  on  gelatin,  this  yeast 
only  produces  along  its  line  of  inoculation  a  small  number  of  cells 
which  remain  colorless ;  with  the  exception  of  those  on  the  surface, 
no  liquefaction  is  produced.  The  cells  die  in  5  minutes  at  50°  C. 

1  Yabe,  K.     On  two  new  kinds  of  red  yeast.     Bull,  of  the  Imp.  University 
of  Tokyo,  1902.. 

2  Yabe.    See  reference  for  S.  japonicus. 


PSEUDOSACCHAROMYCES  APICULATUS  323 

TORULA  BOGORIENSIS  RUBRA.     De  Kruyff 

This  is  a  yeast  which  was  isolated  from  the  soil  of  Java  by  Kruyff.1 
It  possesses  the  very  interesting  property  of  fixing  atmospheric  nitro- 
gen. It  does  not  ferment  any  sugar,  secretes  amylase,  lipase  and 
sucrase  and  forms  round  colonies  which  have  a  reddish  tinge  in  the  cen- 
ter. Other  rose-colored  yeasts  have  been  described  as  Saccharomyces 
roseus  (Frank)  Zopf  and  the  Torula  roseaca  Van  Hest. 

TORULA  RUBEFACIENS.    Grosbusch2 

The  cells  are  round  or  elliptical  (3. 7-2-6  JJL).  There  is  abundant 
development  in  beer  wort  with  great  pigment  production.  This 
is  red  and  soluble  in  water  and  exhales  a  fruity  odor.  Giant  colonies 
on  wort  gelatin  are  strongly  colored  red.  Gelatin  is  rapidly  liquefied. 
On  potato,  the  yeast  gives  a  red  colony.  The  production  of  pigment 
is  influenced  by  the  kind  of  sugar  in  which  the  yeast  finds  itself,  fer- 
mentable sugars  favoring  this  action.  The  concentration  of  the  sugar 
and  the  amount  of  acid  are  also  determining  factors.  The  yeast  fer- 
ments levulose  and  dextrose,  acts  less  strongly  on  saccharose  and  a 
little  on  galactose.  Ando,3  in  studying  some  red  yeasts  isolated  from 
breweries  which  were  probably  Torula,  found  that  the  color  did  not 
depend  upon  the  nutrient  medium.  The  red  pigment  was  found  to 
have  intimate  connection  with  the  life  of  the  yeasts.  In  this  case  it 
was  regarded  as  an  indication  of  life. 

Genus  II.    Pseudosaccharomyces.     Klocker 

HANSENIA.    Zikes 

The  cells  are  usually  supplied  at  one  or  both  ends  with  little 
points  like  those  on  lemons. 

PSEUDOSACCHAROMYCES  APICULATUS.     Klocker 
Syn.:  SACCHAROMYCES  APICULATUS.      Reess.     Hansen 

It  has  been  stated  that  the  yeast  under  the  name  of  Saccharomyces 
apiculatus  and  described  by  Rees  and  Hansen  represents  not  a  species 

1  De   Kruyff,   E.     Torula   Bogoriensis  rubra.     Ann.   Jard.   bot.    Buitenzorg. 
3,  1909. 

2  Grosbusch,  T.    Ueber  eine  farblose,  stark  roten  Farbstoff  erzeugende  Torula. 
Cent.  Bakt.  42  (1915),  625-638. 

3  Ando,   K.     On  red  yeasts.     Original  Communications  Eighth  International 
Congress  of  Applied  Chemistry,  14  (1912),  7-12. 


324    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

but  a  group.  Those  which  form  spores  are  classed  as  Saccharomycetes 
and  those  which  form  no  ascospores  are  called  Pseudosaccharomycetes. 

Saccharomyces  apiculatus  described  by  Reess  and  Hansen  is  a  top 
yeast  which  causes  active  fermentation  in  dextrose  but  does  not  take 
it  very  far.  After  three  months,  according  to  Hansen,  only  3  per 
cent  of  alcohol  is  formed.  In  beer  wort,  only  1  per  cent  of  alcohol 
is  formed.  There  is  no  fermentation  of  maltose  nor  inversion  of 
saccharose. 

Klocker  found  this  species  in  garden  soil  at  Carlsberg.  On  wort  at 
25°  C.,  it  has  lemon  or  ellipsoideus  shaped  cells  (5-10  /z  long).  The 
temperature  limits  for  growth  are  36-37°  C.  and  0.5-3.5°  C.  It  fer- 
ments dextrose,  levulose,  d-mannose  and  liquefies  gelatin. 

PSEUDOSACCHAROMYCES  APICULATUS  PARASITICUS. 

Klocker 

SACCHAROMYCES  APICULATUS  PARASITICUS.    Lindner 

Lindner  discovered  this  yeast  in  1895  in  the  body  of  an  Homop- 
tera  Aspidiotus  Nerii  and  also  on  the  laurel,  ivy,  myrtle,  etc.  (Fig. 
145-E.)  It  is  probably  from  these  plants  that  it  gets  into  the  bodies 
of  insects.  This  species  has  the  identical  characteristics  of  Saccharo- 

myces apiculatus.  No  formation 
of  ascospores  has  been  noticed. 
Saccharomyces  apiculatus  parasi- 
ticus  is  transmitted  by  the  eggs 
and  finally  enters  the  larvae  to 
penetrate  their  uttermost  ex- 
tremities. They  do  not  seem  to 

play  a  pathogenic  role  in  Aspi- 
Fig.  145-E.  -  S.  api^Mm  parasitic.      ^^  and  geem  to  Uve  in  a  sort 


A,    Cells  Enclosed  in  the  Protoplasm  of  the  Aspi-  .  .  . 

diotus  Nerii  cells;  B,  Greatly  magnified  cells  (after    OI      SymblOSlS.        IlllS      yeast     has 

not  been  cultivated.     Hartig  has 

found  an  apiculate  yeast  in  the  blood  of  caterpillars  which  is  iden- 
tical with  that  described  by  Lindner.1  However,  it  differs  in  that  it 
causes  a  fatal  disease  among  caterpillars.  Lindner  believes  that 
this  yeast  gets  into  the  caterpillars  from  ivy  which  is  abundant  in  the 
vicinity  of  Hartig's  laboratory. 

1  Lindner,  P.    Ueber  eine  in  Aspidiotus  Nerii  parasitisch  lebende  Apiculatus 
Hefe.    Cent.  Bakt.  1,  1895. 


PSEUDOSACCHAROMYCES   MULLERI  325 

SACCHAROMYCES  MACROPSIDIS  LANIONIS.    Sulc  l 

This  species  was  found  in  the  pseudovitellius  of  certain  Lecanides 
(Macropsis  Lanio).     They  possess  cells  3/x,  in  length  and  I//,  in  width. 
One   of  the   extremities  is   pointed.     (Fig.    129.)     The 
contents  show  a  nucleus  and  an  alveolar  protoplasm  in 
the  alveoli  in  which  metachromatic  granules  are  found. 
Multiplication    is    accomplished   always  at  the    poles. 
The  buds  are  elliptical  and  of   the  shape  of  an  egg.   Fi 
They  separate  from  the  mother  cell,  attain  their  com- 
plete  development,  and  are  never  observed  in  chains. 
The  Saccharomyces  macropsidis  Lanionis  is  closely  re- 
lated to,  if  not  identical  with,  the  Saccharomyces  apiculatus  parasiticus. 
It  has  not  been  cultivated. 

PSEUDOSACCHAROMYCES  AUSTRICUS.    Klocker 

On  must  at  25°  C.,  the  cells  are  ellipsoidal  and  4  to  5  jit  long.  The 
temperature  limits  for  growth  are  35-36°  C.  and  0.5-3.5°  C.  It  fer- 
ments dextrose,  levulose  and  d-mannose.  Gelatin  is  liquefied.  It 
was  found  in  soil  from  the  Austrian  Alps. 

PSEUDOSACCHAROMYCES  AFRICANUS.     Klocker 

On  beer  wort  at  25°  C. ,  the  cells  are  elongated  or  lemon  shaped 
(7-12  microns  in  length) .  The  minimum  temperature  limits  for  growth 
are  36-37°  C.  It  ferments  dextrose,  levulose,  d-mannose  and  maltose 
very  feebly.  It  was  found  in  soil  from  Algeria. 

PSEUDOSACCHAROMYCES   CORTICI.     Klocker 

This  yeast  has  lemon-shaped  cells  on  beer  wort  at  25°  C.  (6-11  At 
in  length).  The  temperature  limits  for  growth  are  36-37°  C.  and 
0.5-3.5°  C.  It  ferments  dextrose,  levulose,  d-mannose  and  maltose 
very  feebly.  Gelatin  is  liquefied.  It  was  secured  from  various  trees 
about  Copenhagen. 

PSEUDOSACCHAROMYCES  MULLERI.     Klocker 

On  beer  wort  at  25°  C.  the  cells  are  small  and  shaped  like  lemons 
or  ellipsoidal  (4-6 ju  in  length).  The  temperature  limits  for  growth 
are  35-36°  C.  and  0.5-3.5°  C.  It  ferments  dextrose,  levulose  and  d- 
mannose  and  liquefies  gelatin.  It  was  found  in  soil  from  Java. 

1  Sulc,  K.  Pseudovitellius  und  ahnliche  Gewerbe  der  Homopteren  sind  wohn- 
statten  symbiotischer  Saccharomyceten.  Sitzungsberichte  der  Konig.  Bohm. 
Gesellsch.  der  Wissenschaften  in  Prag.  March  30,  1910. 


326    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 


PSEUDOSACCHAROMYCES  LINDNERI.     Klocker 

On  beer  wort  at  25°  C. ,  the  cells  are  small  and  either  lemon  shaped 
or  ellipsoidal.  The  temperature  limits  for  growth  are  36-37°  C.  and 
6-8°  C.  It  ferments  dextrose,  levulose  and  d-mannose.  It  was  found 
in  soil  from  Java. 


PSEUDOSACCHAROMYCES   GERMANII.     Klocker 

On  beer  wort  at  25°  C.,  the  cells  are  lemon  shaped  (5-8 /z  long). 
The  temperature  limits  for  growth  are  36-37°  C.  and  6-8°  C.  It  fer- 
ments dextrose,  levulose  and  d-mannose  and  liquefies  gelatin.  It 
was  found  in  soil. 


PSEUDOSACCHAROMYCES  JENSENH.    Klocker 

On  wort  at  25°  C.,  the  cells  are  small  and  elliptical,  resembling  the 
shape  of  lemons  (2-5  ju,  long).  The  temperature  limits  for  growth 
are  5-6.3°  C.  and  37-38°  C.  It  ferments  dextrose,  levulose,  d-mannose, 
saccharose  and  maltose  very  feebly.  Gelatin  is  liquefied.  It  was  iso- 
lated from  Java  soil. 

PSEUDOSACCHAROMYCES  MALAIANUS.    Klocker 

On  gelatin  at  25°  C.,  the  cells  are  shaped  like  lemons  or  sausages. 
The  limits  of  temperature  for  growth  are  36-37°  C.  and  0°-8°  C.  It 
ferments  dextrose,  levulose,  d-mannose,  saccharose,  and  maltose 
very  feebly.  Gelatin  is  not  liquefied.  It  was  isolated  from  soil  from 
Java. 

PSEUDOSACCHAROMYCES  LAFARI.     Klocker 

On  beer  wort  at  25°  C.,  the  cells  are  elongated,  in  the  shape  of 
lemons  or  ellipsoidal.  The  temperature  limits  are  36-37°  C.  and  6-8°  C. 
It  ferments  dextrose,  levulose,  d-mannose,  saccharose  and  has  feeble 
action  on  maltose.  Gelatin  is  liquefied. 

PSEUDOSACCHAROMYCES  WILLH.     Klocker 

On  beer  wort  at  25°  C.,  the  cells  are  ellipsoidal  or  elongated  and 
lemon  shaped.  They  are  small  (4-10 ju  in  length).  The  temperature 
limits  for  growth  are  37.5-38.5  and  6-8°  C.  It  ferments  dextrose, 
levulose,  d-mannose,  saccharose  and  maltose  very  feebly.  Gelatin 
is  liquefied.  It  was  found  in  the  soil  of  St.  Thomas. 


PSEUDOSACCHAROMYCES  OF  WILL  327 

PSEUDOSACCHAROMYCES  ANTILLARUM.     Klocker 

On  beer  wort  at  25°  C.,  the  cells  are  small  and  lemon-shaped  or 
elliptical  5  to  12  ju-  long.  The  limits  of  temperature  for  growth  are 
37°-38°C.  and  3^°  C.  It  ferments  dextrose,  levulose,  d-mannose, 
saccharose  and  maltose  feebly.  Gelatin  is  liquefied.  This  yeast  was 
isolated  from  soil  from  St.  Thomas. 

PSEUDOSACCHAROMYCES  OCCIDENTALS.     Klocker 

On  beer  wort  at  25°  C.,  this  species  possesses  lemon-shaped  cells 
(6  to  10  ju  long).  The  limits  of  temperature  for  growth  are  39-40°  C. 
and  3  to  6°  C.  It  ferments  dextrose,  levulose,  d-mannose  and  sac- 
charose and  acts  feebly  on  maltose.  It  liquefies  gelatin.  It  was  iso- 
lated from  soil  from  St.  Croix. 

PSEUDOSACCHAROMYCES  SAUTRANZENSIS.    Klocker 

The  cells  of  this  yeast  are  elliptical  or  lemon  shaped  on  beer  wort 
at  25°  C.  They  are  from  6  to  10  ju-  long.  The  temperature  limits 
for  growth  are  37-38°  C.  and  3  to  6°  C.  It  ferments  dextrose,  levulose, 
d-mannose  and  maltose  very  feebly.  Gelatin  is  liquefied.  It  was 
isolated  from  soil  from  St.  Croix. 

PSEUDOSACCHAROMYCES  INDICUS.    Klocker 

On  wort  at  25°  C. ,  the  cells  are  lemon  shaped  or  elliptical.  They 
may  be  sausage  shaped  (3-7 jj,  long).  The  temperature  limits  for 
growth  are  37-38°  C.  and  3-4°  C.  It  ferments  dextrose,  levulose, 
d-mannose,  saccharose  and  maltose  very  feebly.  It  liquefies  gelatin. 

PSEUDOSACCHAROMYCES   OF  WILL 

Will  has  isolated  four  species  of  yeasts  from  different  sources  none 
of  which  form  ascospores.  The  shape  of  these  yeasts  is  quite  vari- 
able. The  lemon-shaped  cell  with  points  may  disappear  and  the  cells 
assume  the  spherical  shape.  In  other  cases  the  cells  may  become 
spindle-shaped.  Some  of  the  cells  are  filiform  while  others  are  sausage 
shaped.  The  size  of  the  cells  is  also  quite  variable.  They  vary  be- 
tween 5  and  6/z  in  length.  They -may  be  distinguished  from  each 
other  by  their  scums.  Two  of  them  (Nos.  4  and  7)  have  a  well-de- 
veloped scum  while  the  other  two  (Nos.  1  and  3)  form  only  a  ring. 

The  giant  colonies  are  characteristic  in  appearance;  those  for 
yeasts  1  and  3  spread  out  on  the  surface  while  those  for  yeasts  4  and  7 
are  cup-shaped.  Yeasts  4  and  7  liquefy  gelatin  more  quickly  than  the 


328    NON-SACCHAROMYCETES   OR  DOUBTFUL  YEASTS 

other  two  yeasts  and  yeast  4  more  quickly  than  7.  These  four  yeasts 
ferment  dextrose  and  levulose.  The  fermentation  continues  for  a 
long  time  with  yeasts  4  and  7,  longer  than  with  the  other  two.  The 
temperature  limits  for  budding  for  these  four  yeasts  are  below  4°  C. 
and  34r-35°  C.  Yeasts  1  and  3  are  more  resistant  to  alcohol  (ethyl) 
than  the  other  two.  Will  considers  yeasts  1  and  2  as  two  varieties 
of  the  same  species  which  he  designates  under  the  name  of  Pseudosac- 
charomyces  cerevisiae  and  yeasts  4  and  7  as  varieties  of  another  species 
to  which  he  gives  the  name  of  Pseudosaccharomyces  vini. 


TORULA  NIGRA.     Marpmann 1 
Syn.:  SACCHAROMYCES  NIGER.     Marpmann 

This  species  was  isolated  from  milk  by  Marpmann.  It  was  re- 
garded by  this  author  as  related  to  P.  membranaefadens.  The  cells 
are  round  or  oval  (1.5  to  3.0 ju  in  diameter).  In  sugar  solutions,  no 
mycelium  is  produced.  On  gelatin  as  in  other  substrates,  black 
colonies  are  formed.  This  yeast  does  not  seem  to  utilize  saccharose 
and  lactose  but  it  uses  a  small  quantity  of  dextrose.  It  seems  to  se- 
crete either  maltose,  lactase,  amylase,  inulase,  or  invertase.  Hansen2 
has  shown  that  this  yeast  does  not  form  ascospores  and  consequently 
does  not  resemble  P.  membranaefadens.  It  is  related  to  the  genus 
Dematium.  Guilliermond  3  has  confirmed  the  opinion  of  Hansen  and 
shown  that  this  species  possesses  characteristics  which  class  it  with 
the  Dematium.  He  has  shown  that  on  carrot  it  produces,  at  the  end 
of  24  hours,  a  sticky  mass  composed  of  oval,  slightly  elongated  cells, 
clothed  with  a  sort  of  mucus  which  contains  black  particles.  These 
are  without  doubt  the  black  pigment  seen  in  cultures.  After  a  few 
days  there  is  formed  at  the  less  moist  parts  of  the  carrot  culture  a 
very  slender  mycelium,  which  rises  from  the  black  mass  of  the  yeasts. 
According  to  the  investigations  of  Guilliermond  the  yeasts  of  this 
fungus  include  only  a  single  nucleus  and  have  a  structure  analogous 
to  that  of  true  yeasts,  but  the  units  of  the  mycelium  may  enclose 
many  nuclei. 

Hansen  has  observed  two  black  Torula  related  to  Torula  nigra. 
Lindner  has  also  described  a  black  Torula  cultured'  in  Koch's  labora- 

1  Marpmann,  G.     Cent,  allgemeine*  Gewidlets  Richard  Landw.     Jahrbucher, 
1891. 

2  Hansen,  E.  C.     Ueber  rot  und  schwarzgefarbte  Sprosspilze.     Allg.  Grauer- 
und  Hopfenzeitung.     1887. 

3  Guilliermond,    A.      Recherches    cytologiques    sur    les    levures    et    quelques 
moisissures  a  forme  levures.     Thesis  for  the  Doctorate  at  the  Sorbonne.     Rev. 
generate  Botanique,  15,  1903. 


TORULA  NIGRA  329 

tory  which  formed  black  yeast  bodies  and  finally  a  deep  green  myce- 
lium.    This  seemed  to  be  related  to  Marpmann's  yeast. 

Other  black  yeasts  have  been  mentioned  by  Marpmann  under  the 
name  of  Schizosaccharomyces  niger  and  Musa.  These  are  not  well 
known  and  seem  to  be  related  to  Dematium  more  than  yeasts.  They 
possess  a  complex  mycelium.  In  all  cases  they  have  been  improperly 
called  Schizosaccharomyces  for  they  are  budding  yeasts  which  possess 
none  of  the  characteristics  of  the  Schizosaccharomyces.  There  have 
been  described  may  species  which  form  a  yellow  and  gray  pigment. 
These  are  too  insufficiently  known  to  be  mentioned  here.  Saccharo- 
myces  sphoericus  might  be  mentioned.  Browne  has  described  a  Mo- 
nilia  nigra,  the  characteristics  of  which  are  given  later  in  this  book. 

In  a  recent  investigation,  Will  isolated  three  forms  of  black  yeasts 
which  he  regards  as  varieties  of  the  same  species.  The  three  forms 
have  a  typical  mycelium  and  a  budding  mycelium.  The  mycelium 
is  a  little  branched  and  forms  conidia  which  are  ellipsoidal  or  spherical 
with  thick  walls.  These  multiply  by  budding,  forming  new  yeasts  or 
producing  another  mycelium. 

In  liquid  media  the  three  forms  of  yeast  develop  on  the  surface 
of  liquid  cultures  and  on  the  walls  of  the  container  with  a  typical 
mycelium.  In  the  bottom  of  the  flask  there  develops  a  flocculent 
sediment  made  up  of  yeast  cells  and  mycelium.  A  ring  develops 
around  the  side  of  the  container  and  is  cartilaginous,  and  a  deep 
black  in  color.  The  scum  is  more  or  less  colored  a  dark  green;  it  is 
thick  and  quite  tough.  The  giant  colonies  are  a  deep  black.  They 
are  made  up  of  a  mycelium  and  budding  cells.  Growth  for  the  three 
species  stops  at  35°  C.  The  three  varieties  are  killed  in  30  minutes 
at  48°  C.  They  do  not  develop  in  media  with  4  per  cent  of  alcohol 
added.  They  are  slightly  resistant  to  alcohol.  No  fermentations 
are  induced. 

Form  I.  The  budding  cells  are  ellipsoidal,  elongated  and  some- 
times apiculate  (3. 9-8. 5  /*).  Usually  they  are  isolated  but  may  be 
grouped,  three  or  four  cells  being  in  a  group.  Some  of  the  cells  are 
giant  cells. 

Form  II.  The  budding  cells  are  oval,  sometimes  sausage-shaped 
(3.9-7. 6 /z).  They  are  sometimes  grouped. 

Form  III.  The  budding  cells  are  spherical,  sometimes  ellipsoidal 
or  sausage-shaped.  The  mycelial  structure  is  less  developed  than  in 
the  two  preceding  forms.  Will  found  no  relation  between  these  yeasts 
and  Cladosporium  herbarum.  On  the  other  hand  he  does  recognize 
relationships  between  these  yeasts  and  Dematium  but  they  are  separated 
by  other  characteristics. 


330    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 


TORULA  FROM    "SOYA"   MASH.     Kita 

Kita1  examined  different  "soya  "  mashes  and  found  a  yeast  which  was 
much  like  Saccharomyces  soya,  Saito,2  with  the  exception  that  no  asco- 
spores  were  found.  Kita  inoculated  a  sample  of  the  mash  into  "soya  " 
decoction  containing  salt.  This  was  eventually  plated  out  on  "koji " 
gelatin  to  which  10  per  cent  of  salt  was  added.  Lindner's  droplet 
method  was  finally  employed  for  getting  pure  cultures. 

The  cells  were  usually  round,  sometimes  elliptical  with  thick 
walls  which  were  easily  visible  under  the  microscope.  The  plasma 
was  wavy.  Vacuoles  were  seldom  seen.  The  size  of  the  cells  in 
"koji"  decoction  was  4.5-8 /i. 

The  colonies  on  "koji"  extract-gelatin-agar  were  round  or  star- 
shaped,  colored  yellow,  elevated  in  the  middle  with  a  smooth  periph- 
ery. Giant  colonies  on  the  same  medium  are  yellow,  with  a  sunken 
center,  granular  surface  and  wavy  periphery.  Streak  cultures  are 
moist,  yellow,  granular  and  with  wavy  edges.  In  "koji  "  extract  to 
which  10  per  cent  of  salt  has  been  added,  growth  is  luxuriant.  A 
ring  is  formed  and  the  medium  seems  to  contain  suspended  floes.  It 
ferments  glucose,  maltose,  but  not  galactose,  sucrose,  lactose,  ramnose 
nor  arabinose.  The  optimum  temperature  for  growth  and  fermenta- 
tion is  about  28°  C.  No  endospores  are  formed  by  young  cells  on 
the  plaster  block.  There  seem  to  be  no  described  species  of  Torula 
which  agree  with  the  characters  of  this  one. 

Genus  III.    Mycoderma.3    Persoon 

Under  this  name  are  grouped  a  number  of  yeasts  which  vegetate 
normally  in  contact  with  air  and  which  form  a  scum  but  do  not  cause 
an  alcoholic  fermentation.  At  the  beginning  of  the  culture  period, 
there  is  formed  a  folded  sc  m  filled  with  air  bubbles.  Ordinarily 
long  cells,  budding  at  the  ends  with  a  transparent  protoplasm  with 
one  or  more  refractive  granules  at  both  poles,  are  present.  The 
Mycoderma,  on  the  whole,  seem  to  possess  the  characteristics  of  the 
fourth  group  of  the  Saccharomycetes  (Pichia  and  Willia)  and  are 
perhaps  asporogenic  forms  of  the  latter.  Some  of  them  are  pig- 
ment ed.  The  Mycoderma  are  very  widespread  in  air  and  live  es- 
pecially on  solutions  containing  alcohol. 

1  Kita,    V.    G.      Haupthefe    der    sojamaische.      Orig.    Communications    8th 
International  Congress  of  Applied  Chem.  14  (1912),  99-106. 

2  Saito,  K.    Cent.  Bakt.  Abt.  2,  17,  104,  152. 

3  It  is  well  not  to  confound  the  Mycoderma  with  Mycoderma  aceti  which  is  a 
bacterium. 


MYCODERMA   VINI  331 

MYCODERMA  CEREVISIAE.     Desm,  Hansen 

Described  by  Hansen  l  after  finding  it  in  the  breweries  of  Copen- 
hagen this  yeast  possesses  cells  of  varied  shapes.  Ordinarily  the  cells 
are  transparent.  Each  cell  usually  contains  one  to  three  small  re- 
fractive granules  (Fig.  146).  On  beer  wort,  this  yeast  produces  a 
dull  gray  scum  frequently  folded.  It  does  not  invert  saccharose  and 
gives  no  fermentation.  On  beer  wort  gelatin,  spots  of  a  gray  color 
are  formed.  Mycoderma  cerevisiae  forms  its  sc  ms  between  2  and  15°  C. 
and  up  to  33°  C.  It  may  cause  considerable  damage  in  beer  which 
it  attacks. 

Hansen  was  the  first  to  show  that  this  yeast  is  not  a  well-defined 
species  but  rather  a  group  of  species  which  has  been  confirmed  later 
by  Lasche*.  This  author  describes  four 
species  which  are  distinguished  from  the 
yeast  described  by  Hansen  in  that  in  beer 
wort,  they  produce  alcohol,  one  0.26  per 
cent  by  volume,  two  others  0.79  per  cent 
and  a  third  0.51  per  cent.  All  of  these 
cause  disease  in  beer.  Lafar  has  dis- 
covered another  Mycoderma  very  closely 
related  to  the  latter  which  forms  a  scum 
quite  closely  related  to  that  formed  by  Fig.  1 46.  —  Mycoderma  cerevisiae 
Mycoderma*  cerevisiae  and  which  gives  ^aS^inCopenh''eel1 
acetic  acid. 

H.  Leberle  and  Will  have  described  two  species  of  Mycoderma 
cerevisiae.  The  first  Mycoderma  cerevisiae,  var.  a  has  cylindrical  cells 
sometimes  elongated  (2-3  ju  wide  and  7-10 ju  long).  The  giant  colonies 
are  very  uniform.  The  temperature  limits  for  vegetative  growth  are: 
7-30°  and  the  optimum  20-25°  C.  This  species  assimilates  only  levu- 
lose.  It  oxidizes  alcohol  quite  energetically  and  assimilates  organic 
acids  easily. 

The  second  Mycoderma  cerevisiae  var.  c,  possesses  oval  cells  or 
cylindrical  cells  (2-4 /i  wide  and  6  to  10 ju  long).  The  temperature 
limits  for  growth  are:  7°  C.  and  30°  C.  The  optimum  is  20-25°  C. 
This  species  assimilated  glucose  and  levulose;  like  variety  a  it  acts 
towards  alcohol  and  organic  acids. 

MYCODERMA  VINI.    Desm 

This  species  has  been  described  by  Seynes,  Wortmann  and  Wino- 
gradsky.  It  presents  some  of  the  characters  of  Mycoderma  cerevisiae. 

1  Hansen,  E.  C.  Levures  alcooliques  ressemblant  a  des  Saccharomyces. 
Comp.  Rend,  des  trav.  du  lab.  de  Carlsberg,  2,  1888. 


332.  NON-SACCHAROMYCETES  OR  DOUBTFUL   YEASTS 

The  cells  are  oval  and  contain  two  vacuoles  filled  with  refractive  gran- 
ules. At  the  beginning  of  their  development  the  cells  are  united  in 
budding  chains.  Later  they  separate.  In  old  cultures,  the  yeast 
takes  irregular  shapes,  some  cells  becoming  angular.  De  Seynes 
thought  that  he  saw  ascospores  in  this  species.  This  work  was  re- 
peated by  Engel,  Reess  and  Cienkowski  but  not  confirmed.  It  is 
then  probable  that  these  pretended  ascospores  were  fat  globules. 
Mycoderma  vini  is  capable  of  changing  the  taste  of  wine.  It  con- 
tributes what  is  called  the  bouquet.  It  oxidizes  alcohol,  changing  it 
into  carbon  dioxide  and  water  with  the  production  of  acid.  It  does 
not  attack  tartaric  very  much  and  citric  not  at  all,  but  destroys  acetic 
acid  and  glycerol. 

According  to  Siefert,1  it  is  necessary  to  distinguish  two  types  of 
Mycoderma  vini:  Mycoderma  vini  I  and  Mycoderma  vini  II.  The 
first  possesses  cells  3  to  10  /i  long  and  from  2  to  4/i  wide.  The 
scum  is  at  first  smooth,  later  folded  and  grayish  in  color.  The  tem- 
perature limits  for  budding  in  wine  with  8  per  cent  of  alcohol 
added,  are  minimum,  5-6°  C.,  optimum,  25-20°  C.  and  maximum,  30°  C. 
This  species  requires  alcohol  for  development  and  attacks  malic  acid. 
In  solutions  containing  4.8  per  cent  of  alcohol  and  malic  acid,  1.52 
per  cent  of  glycerol  is  formed  in  14  weeks.  All  of  the  alcohol  is  de- 
stroyed. In  Austrian  wine,  in  26  days  the  amount  of  glycerol  changes 
from  6.8  per  cent  to  82.  It  forms  9.04  per  cent  of  acetic  acid  and  the 
amount  of  alcohol  changes  from  7.8  to  3.8  per  cent. 

Mycoderma  vini  II  has  temperature  limits  lower  that  those  for 
the  above  yeast:  minimum,  1  to  2°  C.,  optimum,  22°  C.  and  maximum 
28°  C.  to  30°  C.  It  attacks  malic  acid  only  feebly.  In  Pasteur's 
solution  in  a  week  it  gives  0.16  per  cent  of  glycerol.  The  amount  of 
alcohol  is  4.8  to  4.1  per  cent  by  volume.  In  Austrian  white  wine,  after 
26  days  no  increase  in  the  amount  of  glycerol  is  accomplished.  There 
is  formed,  however,  0.64  per  cent  of  acetic  acid.  The  quantity  of 
alcohol  decreases  from  7.8  to  6.8  per  cent  by  volume. 

In  a  recent  investigation,  Gino  de  Rossi  has  shown  that  the 
species  Mycoderma  vini  is  really  made  up  of  a  series  of  distinct  va- 
rieties. By  isolating  the  mycodermic  forms  from  grape  must  or 
wine,  which  had  been  exposed  to  the  air,  this  author  has  been  able  to 
characterize  the  species. 

Mycoderma  vini.  On  grape  must,  the  cells  are  variable  in  form, 
oval,  or  elongated  cylinders  (5.6-9.5  X  2.8-4.8  ju),  united  in  small 
groups  which  branch,  but  which  separate  in  from  4  to  8  days  into 
large  cells  with  from  2  to  3  refractive  granules.  On  gelatin  with  10 

1  Siefert.  Saccharomyces  membranaefaciens.  Ber.  chem.  physiol.  Versuchsst. 
Klosterneuburg,  6,  1899-1900. 


MYCODERMA  HENNEBERG  333 

per  cent  of  grape  must,  the  colonies  are  white  and  round  with  a  plain 
border.  On  wine  or  grape  must,  there  is  a  scum  in  the  beginning. 
The  yeast  gives  no  fermentation  in  must.  It  forms  alcohol  from  wine 
without  noticeably  diminishing  the  acidity.  The  temperature  limits 
are  2°-5°  C.  and  39°  C.,  the  optimum  being  32°-35°  C.  Wine  is 
sterilized  by  heating  for  10  hours  at  50°  C.  and  1  hour  at  55°  C. 
Direct  sunlight  in  June  produced  the  same  results  in  10  hours. 

Mycoderma  duplex.  On  grape  must  or  wine,  the  cells  are  oval  or 
pear  shaped  (3-7.2  X  2-3.6  /A).  After  4  to  8  days,  the  cells  are  oval, 
small,  and  apiculate  with  either  1  or  2  refractive  granules.  Sometimes 
large  oval  or  globular  cells  appear  (5.4  X  10.2ju). 

On  gelatin  with  grape  must,  the  colonies  are  round  with  an  entire 
edge.  On  grape  must  or  wine,  a  white  delicate  scum  is  formed  at  the 
beginning  adhering  to  the  sides  of  the  container.  Finally,  it  breaks 
away  and  falls  to  the  bottom  as  a  fine  deposit. 

There  is  no  fermentation  in  must,  a  slight  diminution  in  the 
amount  of  alcohol  in  wine  and  a  modification  of  the  acidity.  It  is 
able  to  withstand  10  per  cent  of  alcohol  and  2  per  cent  of  tartaric 
acid.  The  temperature  limits  are  5-7°  C.  and  39^40°  C.,  the  optimum 
being  35°  C. 

Wine  containing  this  yeast  is  sterilized  by  10  hours'  heating  at 
48°  C.  and  1  hour's  heating  at  55°  C.  An  exposure  of  8  hours  to  sun- 
light also  destroys  it. 

Mycoderma  tenax.  On  grape  must  or  wine,  the  cells  are  elliptical 
(4.8-8  X  2.8-3.8  M)  and  solidly  united  in  groups  which  branch.  After 
3-8  days,  the  cells  are  round,  or  oval,  with  a  large  refractive  granule. 
On  gelatin  or  grape  must,  the  colonies  are  white  and  round  with  a 
plumose  edge.  On  grape  must  or  wine,  a  delicate  scum  is  formed 
which  clings  to  the  walls  of  the  culture  flask  but  later  falls  to  the 
bottom  of  the  container.  There  is  no  fermentation  in  must,  but  a 
diminution  of  the  alcohol  and  acid  content  of  wine.  It  develops  in 
the  presence  of  4  or  5  per  cent  of  alcohol  and  2  or  3  per  cent  of  tar- 
taric acid.  The  temperature  limits  are  12°  and  32-359  C.,  the  opti- 
mum being  30-32°  C.  Wine  containing  this  yeast  is  sterilized  by  heat- 
ing for  10  hours  at  48°  C.  or  1  hour  at  53°  C.  Exposure  to  direct 
sunlight  for  10  hours  will  kill  the  yeast. 


MYCODERMA  HENNEBERG 

Henneberg  l  mentioned  two  species  of  Mycoderma  which  he  found 
in  brewery  yeasts  and  compressed  yeast.     These  two  species  differ  in 

1  Henneberg,  W.     Zwei  Kahmhefearten  an  abgepresster  Brennereihefe.     Zeit. 
Ges.  Brau.    26,  1903. 


?34    NON-SACCHAROMYCETES  OR  DOUBTFUL   YEASTS 

the  shape  of  their  cells.  One  of  them  produces  filaments  resembling 
the  mycelium  of  Monilia.  Finally  they  may  be  distinguished  by 
their  macroscopic  appearance  in  solid  media  (giant  colonies,  streak 
cultures,  etc.).  In  solutions  of  dextrose  and  levulose,  both  species 
form  a  scum  which  is  filled  with  bubbles  of  carbon  dioxide,  the  cells 
fall  to  the  bottom  of  the  culture  flask  and  induce  a  very  active  fer- 
mentation. Both  species,  like  Willia  anomala,  form  ethyl  ether. 
The  optimum  temperature  for  budding  in  these  species  is  32-41°  C. 
These  yeasts  easily  ferment  dextrose  and  levulose  but  scarcely  act  on 
maltose  and  dextrine,  and  not  at  all  on  lactose,  saccharose,  raffinose, 
and  inuline.  In  dextrose  solutions,  about  37  per  cent  of  alcohol  is 
formed  by  volume.  Both  species  are  able  to  utilize  lactic  acid  as  a 
food;  they  endure  up  to  5  per  cent  of  this  acid.  They  are  also  able 
to  withstand  quite  large  amounts  of  alcohol  (11  per  cent).  The 
alcohol  is  rather  rapidly  oxidized  to  CO2  and  H2O. 

MYCODERMA  CUCUMERINA.     Aderhold 

Discovered  by  Aderhold,1  this  species  lives  in  beer  and  wine  and 
brings  about  certain  undesirable  changes  with  an  acrid  taste.  It 
oxidizes  alcohol  and  lactic  acid  and  produces  from  them  volatile  acids ; 
however,  it  does  not  grow  in  rnore  than  1  per  cent  of  alcohol.  This 
species  may  also  transform  alcohol  into  succinic  acid,  malic  acid  and 
tartaric  acid. 

MYCODERMA  VALIDA.     Leberle-Will 2 

The  cells  are  cylindrical  or  oval  (6-8 ju  long  and  2-4  ju  wide). 
The  temperature  limits  for  growth  are  1-45°  C.,  optimum  20-25°  C. 
This  yeast  assimilates  dextrose  and  levulose  and  oxidizes  ethyl  alcohol 
very  energetically.  It  assimilates  the  organic  acids  very  easily,  es- 
pecially lactic  acid. 

MYCODERMA  GALLICA.     Lerberle-Will 3 

The  cells  of  this  yeast  are  either  oval  or  cylindrical  (7-10  jit  long 
and  2-3 jit  wide).  The  temperature  limits  for  growth  are  7  and  30°  C. 
The  optimum  is  20-25°  C.  This  species  assimilates  dextrose  and  levu- 
lose. It  oxidizes  alcohol  quite  energetically  and  easily  assimilates  the 
organic  acids. 

1  Aderhold,  R.    Arbeiten  der  Botan.  Abteilung  d.  Versuchsstation.  d.  pomolog. 
Inst.  zu  Proskau.     Cent.  Bakt.  5,  1899. 

2  Will,   H.     Beitrage  zur  Kenntniss  der   Gattung   Mycoderma  nach  Unter- 
suchungen  von  Hans  Leberle.    Cent.  Bakt.  28,  1910. 

3  Will,  H.    See  reference  under  Mycoderma  valida. 


SAITO'S  MYCODERMA  335 

MYCODERMA  DECOLORANS.    Will1 

This  yeast  possesses  cylindrical  cells  sometimes  a  little  conical 
with  a  median  constriction  more  or  less  marked.  The  dimensions 
are  variable.  The  temperature  limits  for  growth  are  5  and  42°  C. 
The  optimum  is  25-31°  C.  This  species  oxidizes  alcohol  very  ener- 
getically. The  giant  colonies  on  must  gelatin  are  very  flat,  thin  and 
quite  spread  out.  The  edge  is  often  lobate.  The  center  is  a  little 
concave  with  the  peripheral  portion  lined  with  concentric  bands.  This 
species  causes  a  disease  in  beer  characterized  by  a  decoloration  of  the 
substrate,  an  odor  and  a  musty  taste. 

SAITO'S  MYCODERMA 

Saito2  has  described  four  species  of  yeasts  as  Mycoderma.  One 
isolated  from  "Shiro-koji  "  forms  a  dry  scum  folded,  white  and  thick. 
The  young  cells  are  oval  (4  to  6ju)  with  abundant  protoplasm,  with 
one  or  more  vacuoles  and  one  or  three  fat  globules.  This  yeast  causes 
no  fermentation. 

The  other  two  have  been  encountered  in  "Chinese  yeast "  from 
Corea  along  with  Saccharomyces  Coreanus.  On  sugar  solutions,  one 
forms  a  dry,  thin,  dull  scum.  The  cells  are  ellipsoidal,  often  shaped 
like  a  sausage  (4-8  ju  long  and  4-6  jit  wide)  with  homogeneous  con- 
tents and  provided  with  small  granules.  It  produces  only  a  slow 
liquefaction  of  gelatin  and  a  very  feeble  fermentation.  The  other, 
on  the  surface  of  sugar  solutions,  forms  a  farinaceous,  white  scum. 
The  cells  are  oval  or  spherical  (2-6  jit  in  diameter)  and  possess  an  in- 
terior with  one  or  more  fat  globules.  On  gelatin  streaks,  the  growth 
is  snow  white  and  presents  a  rough  surface.  Liquefaction  is  quite 
rapid.  This  species  produces  a  feeble  fermentation. 

The  fourth  species  was  isolated  from  fermentation  products  of  the 
soy  bean.  It  has  irregularly  shaped  cells,  elongated  or  oval,  much 
like  those  of  Saccharomyces  pastorianus,  with  a  large  vacuole  contain- 
ing refractive  granules.  On  gelatin  plates,  this  yeast  produces  small 
colonies  with  a  moist  appearance  and  with  a  slightly  raised  center. 
The  edge  is  provided  with  fine  indentations.  It  does  not  liquefy  gela- 
tin. On  streak  cultures,  a  grayish  white  deposit  is  produced  and  a 
finely  indented  border  without  liquefaction  of  the  medium.  The 
giant  colony  has  a  white  appearance,  the  surface  being  much  folded 
and  irregular. 

1  Will,  H.    See  reference  under  Mycoderma  valida. 

2  Saito,  K.      Mikrobiol.  Studien  iiber  die   Soya-Bereitung.      Cent.  Bakt.   17, 
1906;    Notes  on  Formosan   Fermentation  Organisms.     The  Botanical  Magazine, 
15,  1902;   Preliminary  Notes  on  Some  Fermentation  Organisms  of  Corea.     The 
Botanical  Magazine,  Tokyo,  23,  1909. 


336    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

On  decoctions  of  "koji,"  this  species  forms  a  thin  scum  smooth, 
shiny  and  dry.  This  is  folded  and  in  old  cultures  becomes  farina- 
ceous. On  beer  wort,  the  scum  forms  slowly  and  has  a  dull  aspect. 
This  yeast  produces  no  fermentation  of  dextrose,  levulose,  d-galactose, 
lactose,  maltose,  saccharose,  melibiose,  mannose  and  raffinose. 

BRUSENDORFS  MYCODERMA 

Isolated  by  Brusendorf  1  from  potatoes  from  the  Danish  Antilles, 
this  species  forms  on  hop  wort  a  thick  resistant  scum  with  a  dry 
appearance.  The  cells  are  oval,  often  slightly  elongated  and  placed  in 
chains  of  three  or  four  individuals.  They  are  5  to  10  JJL  long  and  2 
to  5/x  wide.  The  cultures  often  have  an  acid  odor  due  to  formic 
acid  produced  by  the  yeast. 

SACCHAROMYCES  MYCODERMA  I.    Wehmer2 

This  yeast  was  isolated  from  fermenting  sourkraut  along  with 
Saccharomyces  brassicae  I  and  II.  The  cells  are  always  small  (3.6 
to  5  jit)  with  almost  always  a  refractive  granule  of  variable  size.  They 
provoke  no  fermentation.  On  cabbage  decoction  the  scum  is  white, 
folded  and  tenacious.  On  gelatin,  with  cabbage  decoction  added, 
this  yeast  forms  a  fine  sediment,  white  in  color.  This  species  de- 
stroys lactic  acid  energetically. 

SACCHAROMYCES  MYCODERMA  II.    Wehmer 

Wehmer3  isolated  this  species  from  the  same  source  as  the  pre- 
ceding one.  It  has  ellipsoidal  cells,  never  spherical  but  rather  large 
(8.4  —  4.8  X  6ju).  No  fermentation  is  induced.  On  cabbage  de- 
coction the  scum  is  thin  and  a  dull  gray.  In  old  cultures,  it 
becomes  folded.  This  species  quickly  destroys  lactic  acid. 

DUCLAUX'S  YEAST.     (Mycolevure) 

This  yeast  was  discovered  by  DuClaux4  in  Raulin's  solution  ex- 
posed to  the  air,  where  it  appeared  spontaneously.  It  develops  with 
a  regular  scum  which  is  folded  when  it  lacks  space  to  spread  out. 
Under  such  conditions,  it  becomes  very  thick.  The  scum  is  formed  of 
oval  cells  more  or  less  granular.  They  are  sometimes  as  large  as 

1  Brusendorf.    Ein  Ameinsaure  bildende  Mycoderma.    Cent.  Bakt.  23,  1909. 

2  Wehmer,  C.    Untersuch.  iiber  Sauerkrautgarung.    Cent.  Bakt.  14,  1905. 

3  Wehmer,  C.     See  reference  for  Saccharomyces  mycoderma  I. 

4  DuClaux,  E.     Traite  de   Microbiologie.     Fermentation  alcoolique.     Vol.  3. 
Masson  and  Co.,  Paris,  1900. 


MYCODERMA  LEBENIS  337 

ordinary  yeasts  but  usually  smaller.  The  cells  are  rarely  united  one 
to  the  other  and  are  only  grouped  two  by  two.  This  yeast  is  a  strong 
oxidizer.  It  oxidizes  sugar  to  carbon  dioxide  and  water.  When  in- 
troduced into  a  flask  of  sugar  media  which  are  easily  aerated,  the  al- 
coholic fermentation  is  set  up,  but  not  as  much  sugar  is  transformed  as 
by  ordinary  yeasts.  It  does  not  form  more  than  3  per  cent  alcohol. 

MYCODERMA  FROM  PINEAPPLE.    Kayser l 

This  yeast,  isolated  from  pineapples,  has  elongated  or  elliptical 
cells  (3.5-7  X  2.5-5 /z).  Sometimes  the  cells  are  spherical,  remaining 
attached  in  chains  of  4  or  5  cells  each.  After  24  hours,  in  all  car- 
bohydrate media  slight  acid  with  a  scum  and  ring  is  produced.  The 
cells  formed  are  like  those  formed  in  the  deposit.  In  all  media  in 
which  they  grow,  a  pleasant  ether  odor  is  produced.  The  thermal 
death  point  in  the  moist  state  is  around  53-55°  C.  and  in  the  dry  state 
100-105°  C.  At  these  temperatures,  the  cells  are  killed  in  5  minutes. 
Kayser  has  also  isolated  many  mycoderma  yeasts  from  bananas. 

MYCODERMA  LEBENIS.     Rist  and  Khoury2 

This  species  was  isolated  from  leben.  It  has  cells  about  6-8  /*  long 
and  SJJL  wide,  either  isolated  or  forming  groups  in  mycelium.  In  this 
latter  case,  the  units  are  long  and  thin  (33 /z  long  and  1.5  to  2/z  thick). 
The  ends  are  enlarged,  giving  somewhat  the  appearance  of  bis- 
cuits. The  lateral  buds  give  rise  to  secondary  chains  at  almost  right 
angles.  The  protoplasm  is  finely  granular  with  large  fat  globules. 

On  the  surface  of  plain  gelatin,  the  colonies  are  grayish  white, 
opaque  and  a  little  raised,  with  a  circular  edge  later  indented  with 
stratification  in  concentric  zones.  On  carbohydrate  gelatin,  the 
colonies  are  exclusively  aerobic  and  of  a  greenish  gray  color.  The 
center  is  surrounded  by  an  arborescent  structure.  Stabs  in  lactose 
gelatin  develop  abundantly  on  the  surface  but  slowly  in  the  depths. 
The  culture  resembles  an  inverted  cone.  On  the  surface  of  gelatin,  a 
thin  crust,  dry,  nacreous,  much  firmer  in  the  periphery  than  in  the 
center,  is  formed.  No  liquefaction  of  the  gelatin  is  accomplished.  In 
milk  bouillon,  the  Mycoderma  grows  badly  and  forms  a  thin  scum, 
transparent  and  gray,  which  is  attached  to  the  walls  of  the  culture 
flask.  The  liquid  becomes  cloudy  and  there  is  a  deposit  in  the 
bottom  of  the  flask.  There  is  no  fermentation  of  lactose.  On  grape 
must,  there  is  produced  an  active  fermentation  and  a  thick  scum. 

1  Kayser,  E.    Note  sur  les  ferments  de  1'ananas.    Ann.  Past.  Inst.  5,  1891. 

2  Rist,  E.,  and  Khoury,  J.    Etudes  sur  un  lait  fermente*  comestible,  le  "leben" 
d'Egypte.    Ann.  Past.  Inst.  16,  1902. 


338    NON-SACCHAROMYCETES   OR  DOUBTFUL  YEASTS 

This  species  ferments  maltose,  but  has  no  action  on  saccharose  or 
lactose.  It  seems  to  cooperate  with  Torula  lebenis  in  the  fermenta- 
tion of  milk  but  only  when  it  is  associated  with  Streptococcus  lebenis. 


DOMBROWSKTS  l  MYCODERMA  FROM   MILK 

Mycoderma  lactis  a.  This  yeast  was  encountered  by  Jensen  and 
by  Collau  in  various  milk  products,  particularly  in  butter  from  Fin- 
land. The  cells  are  elongated,  rectangular,  with  rounded  angles; 
sometimes  they  are  slightly  curved.  Besides  these,  one  may  find 
numerous  spherical  cells.  The  cells  enclose  small  droplets  of  fat. 
The  dimensions  of  the  cells  are  quite  variable.  After  96  hours  on  beer 
wort,  the  length  may  be  14.72,  13.0,  9.5,  8.42,  5.5  M  and  their  width, 
4.15,  14.15,  3.7,  3.7,  3.2  ju.  Often  the  cells  may  be  longer  than  27  M- 
At  the  end  of  24  hours,  this  species  forms  on  carbohydrate  liquid 
media,  a  well-developed  scum.  The  wort  becomes  very  cloudy  and 
clears  itself  after  10  days.  In  must  fermentation  is  brought  about 
with  the  escape  of  an  aromatic  odor  like  that  of  ethyl  ether.  After 
five  and  one  half  months,  6  grams  of  alcohol  are  formed  per  100  c.c. 
of  must.  In  milk  at  23-25°  C.,  there  is  no  fermentation. 

On  beer  wort  gelatin  plates,  the  colonies  are  flat  with  a  farina- 
ceous covering  in  the  midst.  In  gelatin  stabs,  development  extends 
down  to  3.5  ccm.  About  the  line  of  inoculation,  one  may  see  extended 
lines  which  decrease  in  length  as  one  goes  toward  the  bottom  of  the 
tube. 

Giant  colonies  have  a  membranous  aspect  with  a  grayish  white 
color.  In  the  center,  a  crateriform  concavity  exists  about  which 
is  a  raised  portion.  The  border  is  finely  fringed  and  possesses  light 
folds.  This  yeast  ferments  only  dextrose.  The  fermentation  is  ac- 
companied with  the  formation  of  ethers. 

Mycoderma  lactis  ft.  Collau  isolated  this  species  in  Copenhagen 
from  a  culture  of  starter  used  in  cream  ripening.  It  is  closely  related 
to  the  Mycoderma  described  above  but  is  distinguished  by  the  size 
of  its  cells  and  by  its  fermenting  ability.  The  appearance  of  the  giant 
colonies  is  also  a  distinguishing  characteristic.  The  cells  may  reach 
12.87;u  in  length  and  3ju  in  width. 

On  beer  wort,  this  yeast  acts  like  the  preceding  one;  however,  it 
has  a  very  feeble  fermenting  ability.  At  the  end  of  five  months,  only 
4.2  grams  of  alcohol  per  100  c.c.  are  formed. 

The  colonies  on  gelatin  plates  are  much  cut  up  and  suggest  the 
structure  of  molds.  The  cells  are  very  much  elongated,  united  and 

1  Dombrowski,  W.  Die  Hefen  in  Milch  und  Milchprodukten.  Cent.  Bakt. 
28,  1910. 


MYCODERMA   CHEVALIERI 


339 


possessing  short  lateral  buds  at  their  points  of  contact.  This  gives 
them  the  appearance  of  the  mycelium  of  Dematium.  Giant  t  colonies 
show  less  development  than  those  of  Mycoderma  lactis  a.  They  are 
membranous  and  possess  a  concavity  surrounded  by  an  elevated 
portion.  The  edge  is  lightly  folded  and  indented.  The  colony  pos- 
sesses a  superficial  crust  of  a  whitish  color. 

Other  Mycoderma  have  been  mentioned  but  they  are  less  known. 
Among  them  may  be  mentioned  Mycoderma  sphaeromyces  (Rothen- 
bach)  which  ferments  dextrine  and  Mycoderma  saprogenes  sake  (Taka- 
hashi)  which  was  found  in  an  alteration  of  sake*. 


f\ 
c~-Q          ^ 

„ 

Fig.  146-A.  —  Mycoderma  Cheva- 

lieri.    Cells    f 
Growth  after 
Guilliermond). 


MYCODERMA  CHEVALIERI.     Guilliermond 

This  species  was  found  along  with 
Saccharomyces  Linderii  in  the  fermenta- 
tion of  an  alcoholic  drink  similar  to  Eng- 
lish ginger  beer.  On  beer  wort  at  25°  C., 
it  develops  rapidly,  forming  a  sedimental 
growth  after  24  hours.  A  scum  is  also 
formed  on  the  surface.  The  scum  ap- 
pears as  little  floating  islands  which  soon 
become  confluent  to  form  a  continuous 
scum  which  adheres  to  the  sides  of  the 
container,  forming  a  marked  ring.  This 

scum  is  very  thin  and  of  a  grayish  yellow  "  7«."  Cells  Trom^'Sedimental 
color.  It  does  not  contain  air  bubbles.  Growth  after  44  Hours  (after 
It  is  very  delicate  and  falls  to  the  bottom 

of  the  culture  flask  when  it  is  dis- 
turbed. Another  re-forms  very 
quickly.  It  has  the  same  characteris- 
tics as  the  scum  formed  by  Zygosac- 
charomyces  Chevalieri. 

When  examined  at  the  end  of  24 
hours,   the  sediment    shows  almost 
constantly  yeasts  isolated  or  united 
two   by  two.     These   are   generally 
small  and  elongated;    rarely  are  they 
short    or    oval.       Their    dimensions 
vary  between  3  and  5ju  wide  and  4 
and  14  JJL  long.     Their  average  size  is 
Fig.  146-B.  —  Mycelial  Formation  in   about    3.96  X  6.21  fJL.     The    contents 
Mycoderma  Chevalieri,  Showing  At-   of  the  cells  are  quite  transparent  with 


tached  Yeast  Structures  (after  Guil- 
liermond). 


.. 
one  or  two  large  vacuoles  less  dis- 


340    NON-SACCHAROMYCETES   OR  DOUBTFUL  YEASTS 

tinct  with  often  many  fat  droplets.  Budding  is  uniquely  accomplished 
at  both  ends  of  the  cells.  The  cells  possess  the  characteristic  ap- 
pearance of  Mycoderma. 

The  scum  is,  at  first,  almost  always  made  up  of  yeast  cells.  These 
have  somewhat  the  same  appearance  as  the  cells  in  the  deposit.  They 
are  rarely  isolated  as  in  the  sediment  and  are  more  often  united  in 
groups  of  from  4  to  8  cells.  Certain  cells  have  a  tendency  to  elongate 
and  may  reach  14  to  20  JJL  in  length. 

After  from  14  days  to  2  months,  a  mycelial  formation  appears  in 
the  sediment.  The  temperature  limits  for  growth  are  5°  C.  and  46- 
57°  C.  On  wort  gelatin  the  giant  colonies  have  a  characteristic  ap- 
pearance. The  center  is  a  creamy  yellow  color  and  is  made  up  of 
fine  reticulations.  The  periphery  is  made  up  of  two  zones;  first,  one 
with  a  white  color  and  thick,  secondly,  one  with  canals  running  out  to 
the  edge  from  this  center.  The  gelatin  is  liquefied.  On  wort  gelatin 
at  20°  C.,  the  colony  is  grayish  white  with  a  dry  appearance.  The 
yeast  causes  a  slight  fermentation  in  beer  wort  and  ferments  sac- 
charose, feebly  dextrose,  energetically  levulose  and  d-mannose. 

MYCODERMA  SP.     Saito 

This  species  forms  on  beer  wort  a  white  thick  scum  which  adheres 
to  the  sides  of  the  containers.  The  cells  are  oval  and  often  curled. 
The  giant  colonies  develop  with  a  thick  gray  vegetation.  The  tem- 
perature limits  for  growth  are  2-3°  C.  and  32-35°  C.  On  "koji" 
decoction,  this  yeast  gives  no  fermentations. 

MYCODERMA  OF  FISCHER  AND  BREBECK 

Fischer  and  Brebeck  l  have  described  a  number  of  Mycoderma  under 
the  generic  name  of  Endoblastoderma  and  Blastoderma.  Such  are  End. 
amycoides  I-IV>  liquefaciens,  and  glucomyces  I-IV  and  Blastoderma 
salminicolor.  The  last  one  is  most  interesting  and  the  best  known. 
It  was  found  in  a  sample  of  sea  water  south  of  the  island  of  Azores. 
The  most  salient  characteristic  of  this  species  is  that  the  cells  form 
long  extensions  at  the  end  of  which  develop  structures  like  conidia. 
These  quite  often  develop  on  the  surface  of  the  liquid  in  contact  with 
air.  When  examined  in  a  hanging  drop,  they  possess  an  excessive 
brilliant  aspect.  This  species  possesses  a  red  pigment. 

Two  other  red  Mycoderma  have  been  described  or  rather  observed 
by  Lasche*.  Mycoderma  humuli,  isolated  from  hop  leaves  and  Myco- 
derma rubrum,  found  in  a  culture  of  contaminated  gelatin. 

1  Fischer,  B.,  and  Brebeck,  C.  Zur  Morphologic,  Biologic  und  Syst.  der  Kahm- 
pilze,  Jena,  1891. 


MYCODERMA  RUGOSA  341 


MYCODERMA  MONOSA.    Anderson1 

"Morphology.  Cells  in  young  cultures  are  elliptical  or  narrowly 
elliptical;  in  old  cultures  cells  are  of  various  forms,  predominantly 
elliptical,  with  numerous  elongated  and 
irregular  forms.  Rows  of  elongated  Q 

cells  in  old  cultures  form  a  false  my-    0  @Q  rt  ^0 
celial  development.     No  true  septation    U  0 

is  observed.     Budding  occurs  from  the 
ends   or  from  shoulders  of  the  young 

cells.     The  size  is  2  X  5.5 /i.  Fig.  UW.-Mycoderma  monosa, 

"Cultural  Characters.     On  all  agar  Anderson. 

Slants     the     Streak     is     Spreading,      dull,     1,  Cells  from  Young  Beer  Wort   Culture; 

°'  2,  Cells  from  Old  Culture. 

white,  flat,  and  becoming  gray  with  age. 

A  heavy  dull  pellicle  is  formed  within  24-48  hours  on  all  liquid  sugar 

mediums  and  on  beer  wort.    There  is  a  villous  growth  along  stab  in 

gelatin. 

"Physiologic  Properties.  Glucose  and  levulose  ferment  readily. 
There  is  no  change  in  litmus  milk.  Sugar  mediums,  with  an  original 
acidity  of  -  1,  become  less  acid  after  1  week.  The  culture  was  isolated 
from  human  feees." 


MYCODERMA  RUGOSA.    Anderson 

Anderson  isolated  this  yeast  from  human  feces  and  characterized 
it  as  follows: 

"Morphology.      Cells    in    young    cultures    are    elliptical,    oblong, 
elongated,  or  somewhat  irregular;   in  old  cultures  the  cells  on  the  sur- 
face  of   the    medium  are    oblong, 
f\     ovate  or   elongated;    beneath  the 
()/}    surface  very  long,  narrow  cells  of 
hyphal  character  are  produced  by 
the  elongation  of  the  bud  at  the 
distal  end  of  another  elongated  cell. 
Fig.   UG-T>.  —  Mycoderma  rugosa,  An-   No    septate   mycelium    is   formed, 
derson.  Budding  in  young  cells  occurs  from 

1,  Budding  Cells  from  Young  Beer  Wort  Culture;     en(J    Qr   SnOulder.      The    size    is    3  X 
2,  Cells  from  Old  Culture. 

6.5  /*. 

"Cultural  Characters.  On  glucose  agar  slant  the  streak  is  white, 
dull,  and  flat,  but  not  spreading;  later  the  surface  becomes  glisten- 
ing and  decidedly  rugose  and  pitted.  Bushy  growths  may  extend 

1  Anderson,  H.  W.     Yeast-like  fungi  of  the  human  intestinal  tract.     Jour. 
Infectious  Diseases,  21  (1917),  341-386. 


342    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

downward  into  the  agar  at  points  along  the  streak.  There  is  a  rapid 
villous  development  in  gelatin  stab  cultures.  A  heavy  pellicle  is 
formed  in  sugar  mediums  and  beer  wort.  Giant  colonies  are  very 
distinctive. 

"Physiologic  Characters.  No  sugars  are  fermented;  there  is  no 
change  in  litmus  milk. 

"This  mycoderma  is  not  distinguishable  from  several  other  species, 
for  example,  M.  cerevisiae,  as  far  as  the  morphologic  and  physiologic 
characters  enumerated  are  concerned.  An  examination  of  photographs 
of  the  giant  colonies  of  various  Mycoderma  species  revealed  the  fact 
that  none  of  these  species  produce  the  peculiar  rugose-pitted  type 
formed  by  the  foregoing  species.  The  production  of  such  type  of 
growth  is  not  confined  to  giant  colonies  on  glucose  agar,  but  is  present 
on  slants  of  glucose  and  beer  wort  agar." 

MYCODERMA  TANNICA.    Asai 

Asai l  has  isolated  a  new  yeast  which  causes  dark  brown  or  black 
spots  on  leather.  The  yeast  grows  in  dextrose  or  levulose  or  other 
sugar  solutions  with  an  ammonium  salt  or  amino  acid  as  the  source 
of  nitrogen.  It  does  not  grow  readily  in  dilute  tannin  solution  but 
when  dextrose  and  amino  acids  are  added  good  growth  takes  place. 
Small  amounts  of  alcohol  and  carbon  dioxide  are  formed. 

MYCODERMA  ACIDIPANI.    Rossi2 

The  cells  are  oval  in  shape  (3.2-6.6  X  2.3-3  M)  and  are  united 
in  branching  groups.  There  are  1  or  2  refractive  granules  in  each 
cell.  On  must  gelatin,  the  cells  are  white,  round,  and  provided  with 
a  slightly  filamentous  border.  In  grape  must,  or  wine,  the  delicate 
scum  is  at  first  compact,  later  thick  and  adherent  to  the  walls.  If 
agitated,  it  falls  to  the  bottom  of  the  container.  There  is  no  fermen- 
tation in  must,  but  a  notable  diminution  in  the  content  of  alcohol 
and  a  marked  increase  in  acidity.  This  yeast  normally  develops  at 
a  concentration  of  from  9  to  10  per  cent  of  alcohol  and  withstands 
from  1  to  2  per  cent  of  tartaric  acid.  The  temperature  limits  are  2-5°  C. 
and  32°  C.  The  optimum  is  between  22°  C.  and  27°  C.  Wine  con- 
taining this  yeast  may  be  sterilized  by  heating  for  10  hours  at  45°  C. 
and  1  hour  at  50°  C.  or  by  an  exposure  of  one  hour  to  direct  sunlight. 

1  Asai,  T.  Physiological  investigation  of  a  new  yeast  which  flourishes  in 
tanning  liquors.  Jour.  College  Science  Imperial  University,  Tokyo,  39,  1-42. 
Journal  of  the  Chemical  Society,  January,  1919. 

1  Rossi,  G.  Micoderma  del  vino.  Le  Stazioni  Sperimentali  Agrarie  Italiene. 
50,  1917. 


MEDUSOMYCES   GISEVII 


343 


Genus  IV.     Medusomyces.     Lindau 

Mycodermic  yeasts  with  a  thick  gelatinous  stratified  scum,  resem- 
bling somewhat  a  medusa. 

MEDUSOMYCES   GISEVII.    Lindau 

This  yeast  was  secured  from  Doctor  Gisevii l  from  the  region  of 
Courland  where  it  is  used  as  a  household  remedy.  It  was  carefully 
studied  by  Lindau.  It  is  easily  cultivated  and  macroscopically  forms 
a  peculiar  covering  on  liquid  media.  Lindau  found  tea  infusion  the 
best  liquid  medium  on  which  to  propagate  it.  This  covering  does 


I; 


Fig.  146-E,— 

1.   Yeast  cells;  2.    Scum  developing  in  a  Culture  Flask. 

not  have  the  appearance  of  ordinary  scums  but  is  made  up  of  an  elastic 
tenacious  mass.  The  liquid  soon  assumes  an  aromatic,  fruity  odor. 
In  older  cultures  the  covering  becomes  a  brownish  yellow  color. 
Microscopic  examination  of  this  covering  shows  the  presence  of  nu- 
merous round  or  elliptical  cells.  The  length  varies  between  5. 5-8.5 /z 

1  Lindau,   G.     Ueber  Medusomyces  Gisevii,  eine  neue  Gattung  und  Art  der 
Hefepilze.     Berichte  deutsch.  Bot.  Ges.  31  (1913),  243-248. 


344    NON-SACCHAROMYCETES  OR  DOUBTFUL  YEASTS 

and  the  width  between  1.5  and  3.8/1.  Budding  takes  place  at  the 
poles.  (See  Fig.  146-E.)  The  formation  of  the  slimy  substance  about 
the  cells  is  not  thoroughly  understood  but  is  probably  intimately 
connected  with  the  outer  cell  wall.  The  peculiar  characteristics  of 
this  yeast  along  with  those  of  the  scum  caused  Lindau  to  propose  a 
special  genus  of  Medusomyces.  He  separates  this  yeast  from  the 
Mycoderma  by  the  characteristics  of  the  scum. 

Lindner  1  examined  some  of  the  material  from  £ourland  which 
was  given  to  him  by  Lindau.  He  found  different  fungi  among 
which  was  Bacterium  xylinum  to  which  he  attributed  the  fermenting 
capacity  of  this  material.  The  presence  of  different  yeasts,  such  as 
Saccharomyces  Ludwigii  and  Schizosaccharomyces  Pombe,  was  also  sug- 
gested. 

1  Lindner,  P.  Die  vermeintliche  neue  Hefe  Medusomyces  Gisevii.  Ber. 
deutsch.  Bot.  Gesell.  31  (1913),  364-368. 


CHAPTER  XII 
PATHOGENIC   YEASTS 

THE  pathogenic  yeasts,  which  do  not  sporulate,  possess  generally 
the  characteristics  of  Torula.     They  may  be  regarded  as  part 
of  this  genus.     However,  Vuillemin  has  created  a  special  generic 
name  for  them,  Cryptococcus.    This  name  is  generally  used  and  so  it 
has  been  retained  for  this  discussion. 

CRYPTOCOCCUS   DEGENERANS.    Vuillemin 
Syn.:  BLASTOMYCES  VITRO  SIMILE  DEGENERANS.     Roncali l 

This  yeast  was  encountered  in  a  ganglion  of  the  armpit  of  a  woman 
attacked  by  a  cancer  and  in  other  tumors.  It  was  both  extra-  and 
intracellular.  In  cancer  the  cells  were  rounded,  rarely  oval,  isolated 
or  in  groups,  without  capsules,  with  homogeneous  contents,  poor  in 
granulations.  In  cultures,  the  cells  are  elliptical,  mixed  with  mycelial 
forms.  In  carbohydrate  solutions,  this  organism  forms  a  scum  com- 
posed of  yeasts  and  mycelium.  In  bouillon,  it  produces  an  abundant 
sediment  made  up  of  cells  and  filaments.  On  gelatin  plates,  the  super- 
ficial colonies  are  irregular,  of  a  grayish  yellow  color;  there  is  no  lique- 
faction. On  gelatin  streaks,  the  growth  is  milky  white.  On  potato, 
the  colonies  are  grayish  white  and  undulated.  The  yeast  does  not 
ferment  saccharose.  It  is  pathogenic  for  guinea  pigs.  Injections  into 
the  peritoneum  cause  death  of  the  animal  in  15  to  30  days. 

CRYPTOCOCCUS  GILCHRISTI.    Vuillemin 

Syn.:    ZYMONEMA  GILCHRISTI.     De  Beurmann  and   Gougerot. — 
BLASTOMYCES  DERMATITIS.     Gilchrist  and  Stokes 

This  yeast  was  found  by  Gilchrist 2  in  a  case  of  scrofular  der- 
matitis and  later  by  Gilchrist  and  Stokes  in  a  case  of  pseudo  lupus 
vulgaris.  It  has  round,  slightly  oval  cells,  20  or  more  ju,  in  diameter, 

1  Roncali.      Die    Blastomyceten    in    den    Adeno-Carcinoman    des    Ovariums. 
Cent.  Bakt.  18,  1895. 

2  Giichrist.      A    case   of   blastomycetic    dermititis   in    man.      Johns   Hopkins 
Hospital  Bulletin.     1896. 

345 


346 


PATHOGENIC   YEASTS 


and  a  thick  membrane.  (Fig.  147.)  In  cultures,  it  has  cells  without 
capsules,  elongated  and  mixed  with  mycelial  filaments. 
No  alcoholic  fermentation  is  brought  about  nor  scum 
formed  on  carbohydrate  media.  It  does  not  liquefy 
gelatin.  It  is  hardly  pathogenic  for  animals. 

Echon-Echeug  have  recently  found  a  yeast  in  the 
serous  secretion  from  a  lesion  in  the  cervical  region, 
simulating  cutaneous  tuberculosis.  The  cells  are 

Fig.  147. -^Crypto-  spherical  (7-16 /z),  united  two  by  two.  The  cultures 
on  Sabourand's  agar  has  yielded  white  colonies  which 
be'come  brownish,  made  up  of  a  mycelium  producing 

cells  like  those  found  in  the  lesions. 


CRYPTOCOCCUS  TOKISHIGEI   (Tokishige).     Vuillemin 

Syn.:  CRYPTOCOCCUS  FARCIMINOSUS,  Rivolta  and  Micellone.  — SACCHA- 
ROMYCES  EQUI,  Marcone.  —  CRYPTOCOCCUS  RIVOLTAE,  Fermi  and 

ArUCh.  —  PARENDOMYCES     OF    RIVOLTA    AND     MICELLONE.        Beur- 

mann  and  Gougerot 

This  yeast  was  discovered  by  Rivolta  and  was  considered  by  this 
author  as  the  parasite  of  epizootic  lymphangitis  or  African  glanders, 
a  communicable  infection  of  horses  and  mules.  Numerous  authors 
have  thought  that  they  cultivated 
this  organism.  Fermi  and  Aruch 
thought  that  they  obtained  it  on 
potato  and  San  Felice  said  that  he 
reproduced  the  disease  by  cultures. 

Marcone  and  Tokishige  were  the 
first  to  obtain  the  development  of 
the  fungus  but  they  were  unable  to 
cultivate  it  in  series.  Tokishige  in 
Japan  has  been  able  to  obtain 
colonies  on  quite  diverse  media,  but 
could  not  produce  the  disease  when 
inoculating  a  horse  with  the  colonies. 
More  recent  studies  by  Negre  and 
Bride  and  Negre  and  Boquet  have 
demonstrated  that  the  parasite  of  this 
disease  is  indeed  a  yeast.  In  a  few  animals,  the  organism  possessed 
the  shape  of  a  yeast.  They  secured  best  growth  of  the  Cryptococcus 
by  sowing  a  drop  of  pus  on  horse  dung  agar  and  covering  it  with 
the  deposit  of  a  maceration  of  lymphatic  ganglions.  The  colonies  are 


Fig.  148.  —  Cryptococcus  Tokishigei. 

1,  Cryptotoccus  in  a  leucocyte.  R,  Round  Form 
of  the  Cryptococcus.  T,  Myoelial  tube  form- 
ing a  bud.  The  bud  is  still  within  the  Leuco- 
cyte. —  2,  Mycelial  Tubes  Formed  by  the 
Budding  of  an  External  Spore  S.  T,  Mycelial 
Tube.  —  3,  External  Spore.  —  4,  Its  Bud.  —  5, 
Chlamydospore  in  a  Mycelial  Tube.  —  6,  Free 
Chlamydospore.  —  7-9,  Free  units  of  an  old 
culture. 


CRYPTOCOCCUS  TOKISHIGEI 


347 


then  transplanted  onto  the  same  medium  as  Sabourand's  medium. 
The  organism  is  easily  cultured  and  develops  rapidly. 

The  most  favorable  temperature  is  37°  C.  At  this  temperature 
the  colonies  on  Sabourand's  agar  have  a  yellow  sandy  appearance. 
They  are  folded  and  have  little  white  points. 

In  the  beginning  the  Cryptococcus  enlarges  and  assumes  a  round 
shape  filled  with  oil  droplets.  It  then  buds  giving  mycelial  tubes  which 
form  lateral  branches. 

On  the  secondary  branches  occur  tertiary  branches.  At  the  end 
of  all  of  the  filaments  small  buds  form,  the  walls  of  which  thicken 
and  the  contents  become  filled  with  fat 
globules.  These  detach  themselves  and 
become  external  spores.  These  are  prob- 
ably the  forms  of  the  organism  which 
multiply  under  the  shape  of  yeasts.  In 
culture,  the  spores  form  new  mycelial 
structures.  The  mycelium  is  also  able 
to  form  at  the  ends  of  the  filaments  a 
small  number  of  segments  with  chlamy- 

dospores   having  a  very  thick  wall    and   Fig.  148-A^— -  Cryptococcus^  To- 
finely  granular  contents.    In  old  cultures, 
the  units  of  the  mycelium  break  off. 

At  times  the  authors  have  noticed  cells 
with  three  or  four  elements  resembling  as- 
cospores.  This  would  tend  to  make  the 
yeast  an  Endomyces.  The  presence  of  ascospores  does  not  seem  to 
be  well  established.  As  to  the  ascospores  described  by  Tokishige,1 
Negre  and  Boquet  have  shown  that  they  were  simply  granular  bodies. 

Cultures  of  the  yeast  inoculated  into  a  horse  by  scarification  of 
the  epidermis  and  subcutaneous  injection  produced  abscesses  and 
finally  a  clinical  history  of  the  natural  disease.  The  Cryptococcus 
appears  in  the  lesions  three  to  four  weeks  after  inoculation.  The 
cells  are  at  first  isolated  and  have  the  shape  of  small  oval  units 
with  thin  walls.  Later  they  take  on  a  double  contour  and  appear 
inside  of  the  leucocytes.  The  serum  from  sick  animals  gives  positive 
reactions  with  cultures  of  the  fungus. 

Boquet  and  Negre 2  have  studied  the  variations  taking  place  in 
the  development  of  Rivolta's  Cryptococcus.  They  found  that  a  mini- 
mum temperature  of  15-18°  C.  caused  this  parasite  to  take  on  a 
mycelian  structure.  At  the  optimum  temperature,  35-36°  C.,  in  liquid 

1  Tokishige.     Ueber  pathenogene  Blastomyceten.     Cent.  Bakt.  19,  1896. 

2  Boquet,  A.,  and  Negre,  L.   Polymorphisme  morphogenique  du  Cryptococcus 
de  Rivolta.     Ann.  Past.  Inst.  33  (1919),  185. 


kishigei,  Cultivated  on  Nutri- 
tive Agar. 

a,  Typical  cells;  b,  c,  Cells  Containing 
Granules  Considered  by  Tokishige  as 
Ascospores;  d.  Free  Granules  Sup- 
' posed  to  have  Sprung  from  the  Cells; 
e,  f,  Transformation  of  the  Cells 
into  Filaments  (after  Tokishige). 


348 


PATHOGENIC  YEASTS 


media,  the  cells  became  round  or  oval  and  were  surrounded  by  a 
double  membrane.  Whether  the  parasite  reproduced  by  budding  or 
not  seemed  to  be  independent  of  aerobiosis. 


CRYPTOCOCCUS  FARCIMINOSUS.     Rivolta  and  Micellone 

Syn.:  SACCHAROMYCES  EQUI.  Marcone.  —  CRYPTOCOCCUS  RIVOLTAE. 
Fermi  and  Aruch.  —  PARENDOMYCES  DE  RIVOLTA  AND  MICELLONE. 
De  Beurmann  and  Gougerot 


This  species  has  been  regarded  as  the  causal  organism 
of  glanders  which  attacks  horses  and  mules.  It  has  round 
or  oval  cells,  sometimes  pointed  at  the  poles,  with 
granular  contents  (Fig.  149).  It  is  easily  cultivated  in 
all  media.  On  potato,  it  produces  a  round,  raised  colony 
with  a  dirty  white  color.  It  scarcely  develops  on  agar. 
Fermi  and  Aruch  l  have  described  in  the  cells  of  this  yeast 
found  in  pus,  globules  which 
they  regarded  as  ascospores. 
These  could  not  be  found  in 
artificial  cultures,  however. 


oo 


Fig.  149.— 
Crypt,  far- 
dminosus. 
Growth  of 
Cells  and 
Ascs  in  the 
Pus  (after 
Fermi  and 
Aruch) . 


CRYPTOCOCCUS  HOMINIS    (Busse). 
Vuillemin 

Syn.:  ATELOSACCHAROMYCES  HOMINIS. 
De  Beurmann  and  Gougerot 

Discovered  by  Busse,2  in  chronic  periostitis 
of  the  tibia,  this  yeast,  in  situ,  possesses  cells 
which  are  round  or  oval,  unites  in  various 
numbers  in  a  substance  with  a  homogeneous 
appearance,  making  a  sort  of  common  cap- 
sule. (Fig.  150,  B.)  In  cultures,  the  same 
shape  is  presented  but  there  is  no  homo-  Fig.  150.  —  Cryptoc.  hominis. 
geneous  substance.  There  is  a  membrane  A.  ow  Culture  on  Prune  Juice.  — 
with  a  double  layer  which  thickens  as  the 
culture  becomes  older.  (Fig.  150,  A.)  It  is 
cultivated  easily  on  all  media  between  15°  and  38°  C.  In  liquid  media 
a  sediment  is  formed  and  on  prune  juice  it  finally  forms  a  scum 
with  a  dirty  gray  color.  In  gelatin  stabs,  the  colonies  are  white  and 

1  Fermi,  C.,  and  Aruch.     Ueber  eine  neue  pathogene  Hefeart  und  iiber  die 
Natur  des  sogennanten  Cryptococcus  farminosus,  Rivolta.     Cent.  Bakt.  17,  1895. 

2  Busse,  Ueber  Saccharomyces  hominis.    Virchow's  Archive,  40,  1895. 


oPfhe 
man 


CRYPTOCOCCUS  LITHOGENES  349 

shiny  but  there  is  no  liquefaction.  On  potato,  the  colonies  soon  unite 
to  form  a  thick  dirty  white  layer.  This  yeast  ferments  dextrose. 
It  is  pathogenic  for  rabbits,  white  rats  and  dogs. 

CRYPTOCOCCUS  LINGUAE-PILOSAE.    Vuillemin 
Syn.  SACCHAROMYCES  LINGUAE-PILOSAE.      Raynard  and  Lucet 

This  yeast  was  discovered  by  Raynard  and  Lucet  in  a  sickness 
called  black  tongue.  Lucet  who  has  studied  this  disease  experi- 
mentally has  shown  that  it  may  not  be  reproduced.  According  to 
Guegen  l  and  Thaon,2  this  yeast  acts  only  in 
association  with  Oospora  lingualis.  There 
seems  to  be  a  sort  of  symbiotic  association 
between  these  two  fungi.  This  yeast  has 
round  or  oval  cells,  often  elongated,  in  which 
the  buds  often  remain  united  to  the  mother 
cell.  This  gives  the  appearance  of  a  pseudo- 
mycelium.  (Fig.  151.)  On  glucose,  levulose,  A.  Ceiis  developed  on  Scum  in 
glycerol  and  especially  potato  decoctions,  ^&^u£^*S> 
or  fruit  decoctions,  after  10  hours  at  37°  C., 

there  is  good  growth.  Later  the  scum  thickens  and  becomes  gray  or 
reddish.  It  may  also  become  folded  with  a  ring.  On  gelatin  this 
yeast  forms  a  mucous  layer,  white,  shining,  with  contours.  On  potato 

it  forms  a  thin  layer,  dry  and  brown.     The 

optimum   temperature    for   budding   is  found 

between  25  and  35°. 

This  yeast  ferments  glucose  and  levulose. 

It  is  pathogenic  for  animals. 
V 

7 

Fig.    \b2.-Cryptococcus  CRYPTOCOCCUS  LITHOGENES.     Vuillemin 

lithogenes. 

2  to  4.  Young  Cells.  -5  to  6.    ty71"'   SACCHAROMYCES    LITHOGENES.     San  FellCC 
Cells  which  have  Undergone  .  . 

a  chalky  Degeneration  (after         This  yeast  was  discovered  by  San  Felice 3 

San  Felice).  ,  J  J  '         . 

in   the   lymphatic   ganglions  of  a   cow  which 

died  from  generalized  carcinoma.  In  the  animal  it  possesses  round 
cells  of  variable  forms  and  dimensions,  sometimes  enclosed  in  a 
calcified  capsule,  with  brilliant  granules  in  the  protoplasm  (Fig.  152). 

1  Guegen,  F.     Sur  Oospora  lingualis  et  Cryptococcus  linguae-pilosae.     Arch. 
de  parasitol.  12,  1909. 

2  Thaon,  P.      Symbiose  de  levure  et  oospora  dans  un  cas  de  langue  noire, 
"oc.  de  Biol.  67,  1909. 

3  San  Felice,  F.     Ueber  eine  fur  Thierpathogene  Sprosspilzart  und  iiber  die 
morph.  Uebereinstimmung  welche  sie  bei  ihren  Vorkommen  in  den  Gesseln  mit 
Krebsascidien  zeigt.    Centr.  f.  Bak.,  V.  XVII.     1895. 


350  PATHOGENIC  YEASTS 

In  a  culture  one  finds  small  cells  with  homogeneous  color  and  with 
fine  membranes  intermingled  with  large  cells  containing  a  refractive 
body  in  their  centers. 

On  glucose  broth  this  species  forms  a  heavy  sediment  and  very 
often  a  scum.  On  gelatin  plates  the  surface  colonies  are  round  like 
pinheads  and  the  deep  colonies  smaller  and  of  a  yellow  color.  In 
gelatin  stabs  this  species  forms  a  white  moist  layer  and  numerous 
colonies.  It  does  not  produce  liquefaction.  On  potato  it  gives  a  fine 
thick  pellicle. 

This  yeast  is  pathogenic  for  guinea  pigs  and  rabbits. 

CRYPTOCOCCUS   GRANULOMATOGENES.    Vuillemin 
Syn.:  SACCHAROMYCES  GRANULOMATOGENES.     San  Felice 

Discovered  by  San  Felice  in  nodules  on  the  lungs  of  pigs,  this 
Cryptococcus  possesses  round  or  slightly  oval  cells  with  a  variable 
edge  with  contents  either  homogeneous  or  vacuolar,  and  with  a  bright 
central  granule. 

On  glucose  broth  it  produces  a  cloudiness  very  rapidly,  and  later 
a  scum.  On  gelatin  plates  it  gives  round  white  colonies.  The  sur- 
face colonies  are  larger.  On  gelatin  stabs  it  produces  a  white  layer 
a  little  raised,  accompanied  along  the  line  of  inoculation  by  a  train 
of  small  yellow  colonies.  No  liquefaction  is  produced.  On  potato  the 
culture  is  elevated  and  slightly  grayish  in  color.  This  yeast  produces 
a  red  pigment  on  honey  and  slices  of  pear.  It  is  slightly  pathogenic 
for  animals. 

CRYPTOCOCCUS  NIGER    (Maffuci  and  Sirleo).     Vuillemin 

This  species  was  discovered  by  Maffuci  and  Sirleo1  in  a  pul- 
monary myxoma  from  a  guinea  pig  inoculated  with  the  liver  of  an 
embryo  coming  from  a  tuberculous  mother.  In  the  myxoma  and  in 
cultures  it  possesses  round  or  oval  cells  with  a  rather  thick  membrane 
and  a  protoplasm  supplied  with  nuclear  bodies.  The  cells  remain 
attached  two  by  two. 

On  liquid  media  the  vegetation  forms  a  white  deposit  and  no  scum; 
on  gelatin  streaks  a  whitish  growth  is  secured;  on  gelatin  stabs  a 
whitish  growth  but  no  liquefaction;  on  potatoes  the  colonies  are 
brown.  This  yeast  produces  alcoholic  fermentation  in  beer  wort  and 
ferments  maltose.  It  develops  between  15  and  40°  C.  It  is  patho- 
genic for  animals,  but  only  for  a  time.  Cultures  sterilized  by  heat 
are  toxic  for  guinea  pigs. 

1  Maffuci  and  Sirleo.  Osservazioni  ed  esperim.  intorno  ad  un  Blastomiceti, 
patogeno  inclusione  dello  stesso  nella  cellula  dei  tessuti  patologici,  Policlinico.  1895. 


CRYPTOCOCCUS  DE  GOTTI  AND  BRAZZOLA        351 


CRYPTOCOCCUS  PLIMMERI.     Costantin1 

This  yeast  was  encountered  by  Plimmer 2  in  a  large  number  of 
cancers.  The  cells  are  round  (4  to  40  ju)  with  a  double  membrane. 
The  cells  may  be  isolated  or  united  to  the  number  of  from  two  to 
sixty. 

On  liquid  media  (2%  glucose  broth  and  1%  tartaric  acid)  it  forms 
a  deposit  at  the  end  of  a  few  days.  On  gelatin  added  to  the  same 
bouillon  the  growth  is  feeble  with  no  liquefaction.  On  agar  added  to 
the  same '  liquid  the  colonies  are  small,  isolated,  slightly  rounded, 
white  in  the  beginning  and  yellow  in  old  cultures.  On  potato  the 
yeast  develops  a  thick  layer,  at  first  white  and  after  a  time  a  brownish 
yellow.  This  yeast  is  pathogenic  for  guinea  pigs  only  on  intraperi- 
toneal  injection. 

CRYPTOCOCCUS   CORSELLH  (Corselli  and  Frisch). 
Neveu-Lemaire 

Isolated  by  Corselli  and  Frisch 3  from  a  sarcoma  in  the  mesenteric 
ganglions  of  a  man,  this  yeast  possesses  black  cells  of  variable  di- 
mensions, slightly  rounded,  and  agglutinated  in  masses.  It  is  easily 
cultivated  on  gelatin,  agar,  dextrose,  broths,  sugar  jellies,  neutral  or 
alkalin.  It  possesses  a  very  feeble  power  of  fermenting,  and  shows  a 
pathogenicity  for  guinea  pigs,  dogs  and  rabbits  in  intraperitoneal 
injections. 

CRYPTOCOCCUS  DE   GOTTI  AND  BRAZZOLA 

Syn.:  ATELOSACCHAROMYCES  DE  GOTTI  AND  BRAZZOLA.     De 
Beurmann  and  Gougerot 

Discovered  by  Gotti  and  Brazzola 4  in  a  myxosarcoma  of  the  nasal 
passages  of  a  horse,  this  yeast  possesses  cells  of  variable  dimensions, 
round  or  slightly  oval,  with  granular  contents  surrounded  by  a  double 
membrane  and  a  mucilaginous  capsule,  sometimes  stratified. 

In  bouillon  it  produces  clumps  and  on  gelatin  stabs  a  train  of 
clumped  masses  with  indented  edges.  On  gelatin  plates  the  colonies 

1  Costantin,  Les  levures  des  animaux,  Bull,  de  la  Soc.  mycologique  de  France, 
v.  XVII.     1901. 

2  Plimmer,   Vorlaiifige  Notiz  iiber  Gewisse  von  Krebse  isolierle  Organismen 
und  deren  pathogene  Wirkung  in  Thieren,  Centr.  f.  Bakt.,  v.  XXV,  1899. 

3  Corselli,    G.,    and    Frisch,    B.    Blastomiceti    pathogene    nelPuomo.     Annali 
d'Igiene  sperim.,  v.  V,  1895,  and  Centr.  f.  Bak.  v.  XVIII,  1895. 

4  Gotti  and  Brazzola,  Sopra  un  caso  di  blastomicosi  nasale  in  una  cavalla. 
Memorie  d.  R.  Ac.  d.  Scienze  di  Bologna,  v.  VI,  1897. 


352  PATHOGENIC  YEASTS 

are  white,  becoming  grayish  yellow  after  a  long  time.  Acid  and 
glucose  gelatin  is  liquefied.  On  glycerin  agar  the  culture  is  creamy 
with  indented  borders.  On  potatoes  the  growth  consists  of  a  thick, 
creamy,  white  layer  which  becomes  brownish  in  old  cultures.  This 
yeast  is  pathogenic  for  guinea  pigs  but  not  for  other  animals. 


CRYPTOCOCCUS  HOMINIS   COSTANTINI  (Costantin). 

Vuillemin 

This  yeast  was  isolated  by  Costantin  l  from  a  cancerous  tumor 
of  the  breast.  It  possesses  round  cells  and  is  distinguished  from 
Crypt,  lithogenes  (San  Felice)  in  that  its  cultures  become  brown  when 
old  and  from  Sacch.  tumefadens  (Busse)  because  its  membranes  never 
become  thick  on  ordinary  media. 


CRYPTOCOCCUS  KLEINII.    Erich  Cohn 

This  species  was  discovered  by  Klein  in  milk,  in  which  it  was  ac- 
companied by  various  pathogenic  bacteria.  It  has  since  been  found 
by  Erich  Cohn.2  The  cells  are  globular,  from  2  to  6  /i  in  size,  with 
homogeneous  contents,  thin  membrane,  and  surrounded  by  a  hyalin 
capsule.  The  capsule  persists  but  becomes  smaller  in  cultures.  This 
yeast  is  easily  cultivated  on  beer  wort  agar.  It  does  not  ferment 
dextrose,  maltose,  or  lactose  nor  liquefy  gelatin. 


CRYPTOCOCCUS  ANOBII.    Escherich 

This  species  was  found  by  Escherich  3  in  the  cells  of  the  intestinal 
wall  of  the  larva  of  Anobium  panicewn.  The  cells 
are  pear  shaped  or  club  shaped,  from  3.5  to  4ju  in 
size,  with  center  provided  with  refractive  granules 

Fig     153  Crypto-   (Fig.  153).    In  culture  it  forms  a  pseudo-mycelium 

coccus  Anobii  (after  made  up  of  cells  shaped  like  a  sausage. 

This  yeast  is  easily  cultivated  in  .01%  of  sac- 
charose, liquid  or  solid  (gelatin  or  agar).  On  gelatin  it  gives  round 
colonies  with  no  liquefaction. 

1  Costantin.   Les  levures   des  animaux.   Bull,   de   Mycologie   de   France,   v. 
XVII,  1901. 

2  Erich  Cohn.  Unters.  liber  eine  neue  thierpathogene  Hefeart  (Hefe  Klein). 
Centr.  f.  Bak.,  v.  XXXI,  1902. 

3  Escherich.    Ueber   das   regelmassige   Vorkommen   von   Sprosspilzen   in   den 
Epithemis  Kasers.  Biol.  Centr.,  v.  XX,  1900. 


CRYPTOCOCCUS  CAVICOLA  353 

CRYPTOCOCCUS  PARASITARIS  (Trabut)  Vuillemin1 
Syn.:  SACCHAROMYCES  PARASITARIS.     Trabut 

Discovered  by  Trabut  on  the  grasshopper  (Acridium  peregrinwri), 
upon  which  it  is  a  parasite,  this  species  has  round  cells,  3  to  4/z, 
provided  with  refractive  droplets.  It  does  not  ferment  dextrose. 

CRYPTOCOCCUS  PSORIARIS.    Rivolta 
Syn.:  SACCHAROMYCES  PSORIASIS.      Cattaneo 

This  yeast,  encountered  by  Rivolta2  in  a  case  of  dermititis,  has 
round  cells  from  28  to  30  jit,  a  double  membrane,  and  is  often  united 
in  chains  of  6  to  8  cells. 

CRYPTOCOCCUS   CAPILLITII.    Vuillemin 
Syn.:  SACCHAROMYCES  CAPILLITII.      Oudemans  and  Pekelharing 

This  yeast  has  been  described  by  Saccardo  3  as  a  spherical  yeast 
from  2.5  to  8/z  in  diameter,  of  a  homogeneous  color,  with  a  thick 
membrane.  It  seems  to  bud.  Blanchard  considers  it  an  Oomycete, 
and  Guegen  thinks  that  it  is  more  closely  related  to  the  Algae.  • 

CRYPTOCOCCUS   OVALIS.     Vuillemin 
Syn.:  SACCHAROMYCES  OVALIS.     Bizzozero 

This  organism  was  discovered  by  Malasses.  The  cells  are  shaped 
like  a  gourd  (3.3  to  3.5  x  2.3  to  2.6/-t).  They  are  made  up  of  a  large 
part  surmounted  by  a  bud.  The  membrane  is  thin  and  the  contents 
include  a  large  brilliant  granule.  Bizzozero  considers  this  parasite 
as  a  yeast  and  gives  the  name  of  Saccharomyces  ovalis  to  it.  It  has 
not  been  secured  in  culture.  It  was  found  in  association  with  the  pre- 
ceding species  and  according  to  Saccardo  may  be  another  form  of  it. 
The  recent  researches  of  Dold4  seem  to  indicate  that  this  organism 
may  not  be  a  yeast  but  a  bacterium. 

CRYPTOCOCCUS   CAVICOLA.     Arthault 

This  species  has  been  found  by  Stephen  Arthault 5  in  a  pulmonary 
cavity.  It  is  cultivated  on  potato  and  on  agar,  giving  moist  colonies, 

1  Guegen,  F.,  Les  champignons  parasites  de  1'homme  et  des  animaux,  These 
agreg.  Pharmacie,  Paris,  1902. 

2  Rivolta,  Parasiti  vegetali,  18-73. 

3  Saccardo,  P.  A.,  Sylloge  fangorum,  v.  VII,  p.  921. 

4  Dold,  H.     On  the  so-called  Bacillus   (Dermatophyton  Malasserzi).     Para- 
sitolog.  3,  1910. 

6  Arthault,  Flore  et  faune  des  cavernes  pulmonaires,  Arch.  d.  Paras.,  1899. 


354  PATHOGENIC  YEASTS 

thick,  with  a  reddish  color.  They  resemble  very  much  the  cultures 
of  Bacillus  prodigiosus.  The  cells  are  small  and  oval,  from  8  to  12  fj, 
long.  This  yeast  is  closely  related  to  Crypt,  glutinis  and  may  per- 
haps be  a  variety  of  it. 

CRYPTOCOCCUS  NEOFORMANS.     San  Felice1 

This  yeast  was  found  by  San  Felice  on  fermenting  fruits.  The 
cells  are  of  variable  dimensions;  some  of  them  have  a  refractive 
granule  in  the  center.  In  the  small  cells  the  center  is  homogene- 
ous. In  the  large  ones  one  finds  a  central  hyaline  part  with  a  very 
refractive  peripheral  ring. 

This  yeast  develops  on  ordinary  substances.  On  gelatin  plates 
the  surface  colonies  are  different  from  those  down  in  the  media. 
The  surface  colonies  are  large  like  the  head  of  a  pin.  They  are 
quite  round  and  form  projections  on  the  surface  of  the  media.  The 
deep  colonies  are  somewhat  spherical  with  a  very  fine  contour.  Gelatin 
is  not  liquefied.  The  colonies  on  agar  have  the  same  appearance. 
Growth  on  gelatin  stabs  develops  as  much  along  the  line  of  inocula- 
tion as  along  the  surface.  There  is  no  liquefaction. 

This  yeast  is  pathogenic  and  causes  tumors  in  animals. 

CRYPTOCOCCUS  OF  CLERC  AND  SARTORY2 

This  species  found  in  chronic  angina  has  elongated  oval  cells,  from 
7  to  10 /-t  by  5 /z  in  size,  isolated  or  in  groups  of  five  or  six.  Budding 
may  take  place  at  one  of  the  ends.  The  cells  easily  take  various 
colors  and  are  not  decolorized  by  the  Gram  method  of  staining.  The 
optimum  temperature  for  budding  is  30°  C.  The  yeast  vegetates 
easily  on  all  the  usual  media  and  especially  on  slices  of  carrot.  It 
does  not  liquefy  gelatin,  coagulates  milk,  and  ferments  lactose  but 
not  galactose.  It  secretes  invertase,  produces  alcoholic  fermentation  of 
dextrose,  but  does  not  hydrolize  starch.  Possessed  of  a  very  feeble 
virulence,  it  is  able  under  certain  conditions,  to  live  in  the  animal 
organism  and  cause  localized  and  curable  lesions. 

BLASTOMYCES  HESSLERI.     Rettger 

Found  by  Rettger3  in  an  abscess  on  the  chin,  this  yeast  develops 
quickly  in  most  of  the  media  at  blood  heat,  but  contrary  to  ordi- 

1  San  Felice,  F.,  Ueber  die  pathogene  Wirkung  der  Sprosspilze,  Zugleich  ein 
Beitrag  zur  Aetiologie  der  bosartigen  Geschwiilste.     Centr.  f.  Bakt.  v.  XVII,  1895. 

2  Clerc,  A.,  and  Sartory,  A.,  Etude  biologique  (Tune  levure  isolee  au  cours 
(Tune  angine  chronique,  C.  R.  de  la  Soc.  de  Biologic,  v.  XIX,  1908. 

3  Rettger,    A.     Contribution   to   the   study   of   pathogenic  yeasts,   Centr.  f. 
Bak.,  v.  XXXVI,  1904. 


CRYPTOCOCCUS  GUILLIERMONDI  355 

nary  yeast,  it  does  not  prefer  an  acid  medium.  It  resembles  Cr. 
Kleinii,  but  differs  by  a  certain  number  of  characteristics  which 
caused  the  author  to  regard  it  as  a  new  species.  This  yeast  is  patho- 
genic to  animals. 

CRYPTOCOCCUS  RUBER.    Vuillemin 
Syn.:  SACCHAROMYCES  RUBER.     Demme 

This  yeast  was  isolated  by  Demme  1  from  cow's  milk,  from  the 
urine  of  a  diabetic  man  and  from  diarrhoeic  stools  of  an  infant  fed 
on  milk.  It  forms  a  red  layer  on  the  sides  of  wooden  pails  which  are 
used  for  milking.  Demme  has  found  it  on  dry  leaves  from  Hayti. 
It  was  studied  later  by  Casagrandi. 

It  is  a  yeast,  round  or  slightly  oval  (Fig.  154),  -with  a  red  or  rasp- 
berry color.  On  gelatin  it  gives  elevated  growth.  At  the  beginning 
the  gelatin  is  not  liquefied,  but  this  change  is 
accomplished  in  about  eight  months,  according 
to  Casagrandi.  According  to  Demme,  Cr.  ruber 
provokes  an  alcoholic  fermentation,  but  this 
property  is  soon  lost  by  continued  culturing  on 
alkalin  media.  Casagrandi  has  not  been  able  to 
observe  it. 

This    yeast    develops    easily  on    glucose    or 
glycerol   agar    and    on    potato.     The   optimum    Fig.  154.  —  Cryptococcus 


temperature  for  budding  is  situated  between  18       ruber , 

spore  (after  Vuillemin). 
and  22  . 

According  to  the  researches  of  the  above-mentioned  investigators 
Cr.  ruber  is  pathogenic.  Introduced  into  the  alimentary  canal  it  pro- 
duces symptoms  of  gastric  enteritis.  Subcutaneous  or  intraperitoneal 
injection  causes  the  formation  of  a  tubercle.  Vuillemin  2  has  shown 
that  the  fungus  isolated  by  Bra  from  different  cancers  is  related  to  Cr. 
ruber,  and  this  latter  seems  to  be  related  to  Cr.  cavicola  of  Stephen 
Arthault. 

CRYPTOCOCCUS  GUILLIERMONDI.   Beauverie  and  Lesieur3 

This  was  isolated  by  Guilliermond  and  Lesieur  from  human  sputum 
during  the  course  of  a  secondary  cancer  of  the  lungs.  This  yeast 

1  Demme,  R.,  Saccharomyces  ruber,  Ann.  de  micrographie,  1889,  and  Annali 
d'Igyene  sperim.,  v.  XVII,  1897. 

2  Vuillemin    P.    Cancer  et  tumeurs  vegetales.  Bull,  des  stances  de  la  Soc.  des 
Sciences  de  Nancy,  1900. 

3  Beauverie,  J-.,  and  Lesieur,  C.     Etude  de  quelques  levures  rencontrees  chez 
Fhomme  dans  certains  exsudates  pathologiques.    Jour,  physiol.  et  pathol.  generale. 
14  (1912)  983-1008. 


356  PATHOGENIC  YEASTS 

exists  as  solitary  cells  or  grouped  in  two;  they  are  round  or  oval  with 
a  thick  membrane  without  a  capsule  (3-5  fJL  X  2.8-4.3  ju). 

In  old  cultures,  the  cells  often  present  abnormal  forms,  either 
sausage  shaped  or  in  chains,  the  cells  of  which  are  capable  of  branching 
into  a  rudimentary  mycelium;  there  are  also  a  number  of  giant  cells 
present.  The  yeast  develops  easily  and  abundantly  in  most  nutritive 
media.  On  carrot,  small,  round  colonies  are  produced  which  become 
confluent  but  irregular  and  viscous.  On  potato,  the  growth  is  feeble 
with  very  small,  white,  dry  colonies. 

On  agar  plates,  vegetation  is  abundant  as  a  viscous  layer  with 
irregular  edges  becoming  yellow.  There  is  no  liquefaction. 

On  fruit  juices,  glucose  or  saccharose  solutions,  there  is  feeble 
development  as  sediment.  No  scum  is  formed.  On  Raulin's  fluid, 
there  is  scant  development  as  a  deposit  with  spherical  cells  (3.5  x  6.4  jut). 


CRYPTOCOCCUS  LESIEURI.    Beauverie  and  Lesieur 

This  yeast  was  isolated  from  an  ulcer  of  the  stomach  during  a 
complication  with  typhoid  fever.  On  beer  wort,  it  has  very  small 
rounded  or  oval  cells  (2-3  /*).  These  may  elongate  and  become 
curled.  The  elongated  cells  are  also  found  united  in  filaments. 
On  beer  wort  agar,  the  yeast  shows  a  white  creamy  colony  with  the 
surface  finely  folded.  It  develops  at  27-37°  C.  On  beer  wort  after  9 
hours  there  is  a  delicate  ring  with  floating  islands  of  scum.  Only 
dextrose  is  fermented.  Animal  inoculation  has  not  yielded  positive 
results. 

CRYPTOCOCCUS  SULFUREUS.     Beauverie  and  Lesieur 

This  yeast  was  isolated  from  a  pharyngeal  exudate  during  an  at- 
tack of  typhoid  fever.  On  carrot,  the  cells  are  elongated,  sometimes 
round  (2-8  M  in  diameter).  This  yeast  develops  well  at  25°  C.-370  C. 
There  is  a  ring  formed  on  beer  wort  after  23  hours.  Dextrose,  lactose 
and  saccharose  are  fermented  slightly.  On  wort  agar,  the  colonies  are 
white  with  a  shiny  surface  and  rounded  edge. 

CRYPTOCOCCUS  ROGERL     Sartory  and  Demanche 

This  yeast  was  isolated  from  a  peritonitis  caused  by  a  perforation 
of  the  stomach.  The  cells  are  long  (3-10  X  2-3  M). 

This  yeast  is  pathogenic  for  the  rabbit  and  guinea  pig.  It  vege- 
tates on  most  of  the  culture  media.  It  was  also  found  by  Beauverie 
and  Lesieur  in  the  pharyngeal  exudate  of  typhoid  fever. 


SACCHAROMYCES  MEMBRANOGENES  357 

CRYPTOCOCCUS  SALMONEUS.    Sartory 

This  species  was  found  by  Sartory  1  with  Oidium  lactis,  in  various 
specimens  of  gastric  contents  in  hyperacidity.  Of  17  of  these  juices 
13  gave  cultures  of  this  yeast.  It  is  a  yeast  quite  closely  related  to 
S.  rosaceus  with  a  beautiful  deep  rose  color  in  which  the  tint  varies 
with  the  temperature  and  the  culture  medium.  The  cells  are  spherical, 
averaging  from  six  to  eight  microns  in  diameter. 

The  optimum  temperature  for  budding  is  situated  between  22 
and  25°  C. ;  however,  the  yeast  develops  from  15  to  34°.  In  this  lat- 
ter case  the  rose  dolor  turns  to  a  pale  tint  and  becomes  very  feeble 
at  39°.  Between  40  and  41°  the  yeast  ceases  to  vegetate. 

This  yeast  forms  a  rose-colored  scum  on  glycerol  broth  at  tempera- 
tures between  15  and  38°.  The  most  favorable  temperature  for  the 
formation  of  scum  is  between  26  and  28°.  The  cells  of  young  scums 
differ  a  little  from  the  cells  in  the  sediment,  but  in  old  ones  they  be- 
come elongated  or  sausage  shaped  and  one  begins  to  notice  structures 
like  a  mycelium.  The  deposit  at  the  bottom  of  the  culture  flask  is 
made  up  almost  entirely  of  spherical  cells  only. 

Cr.  salmoneus  is  easily  cultivated  on  all  solid  media  (gelatin,  agar, 
potato,  carrot).  It  also  develops  easily  on  the  various  liquid  media. 
The  pigment  is  soluble  in  carbon  bisulfide,  benzine,  chloroform,  ethyl 
alcohol,  ether,  acetone  and  is  insoluble  in  methyl  alcohol. 

This  yeast  secretes  invertose  but  does  not  produce  alcoholic  fer- 
mentation. It  is  without  action  on  dextrose,  maltose,  d-galactose, 
starch  or  inulin.  It  precipitates  caseine  in  18  days  but  does  not  pep- 
tonize  the  curd.  It  is  not  pathogenic  for  guinea  pigs,  rabbits  and 
dogs. 

LE  DANTEC'S2  YEAST 

It  was  discovered  in  a  stool  from  Sprue  (chronic  diarrhoea  of  a 
warm  climate)  and  according  to  Le  Dantec  seems  to  be  the  cause  of  this 
sickness.  The  yeast  ferments  glucose  broth  and  does  not  liquefy 
gelatin.  In  aerobic  media  it  grows  especially  like  yeasts;  cultivated 
in  anaerobic  media  it  presents  somewhat  the  form  of  a  mycelium. 

SACCHAROMYCES  MEMBRANOGENES.    Steinhaus3 

This  yeast  was  found  by  Steinhaus  in  a  child  attacked  by  scarlatina 
who  presented  the  phenomena  of  tracheal  stenosis.  Fragments  of 

1  Sartory,  A.,  Cryptococcus  salmoneus,  Bull,  de  la  Soc.  myc.  de  France,  v. 
XXIII,  1907. 

2  Le  Dantec,  A.,  Presence  d'une  levure  dans  la  Sprue,  C.  R.  Soc.  Biol.,  v. 
LXIV,  1908. 

3  Steinhaus,   F.,  Untersuch.   iiber  eine    neue  Menschen  und  Thierpathogene 
Hefeart,  Centr.  f.  Bakt.,  v.  XLIII,  1906. 


358  PATHOGENIC  YEASTS 

membrane  taken  from  the  trachea  gave  on  inoculation  the  culture  of 
this  yeast. 

It  is  a  yeast  with  round  cells,  globular  in  shape,  with  a  reddish 
pigment,  and  sometimes  it  is  very  large.  Often  the  cells  are  pear 
shaped  and  bud.  The  bud  appears  at  one  end  of  the  cell,  which  be- 
comes pointed,  but  the  budding  is  also  accomplished  as  in  other 
yeasts  at  some  other  point  on  the  cell.  The  cells  have  a  double 
wall,  very  fine  and  granular  contents,  with  sometimes  one  or  two 
bright  granules.  In  membranes  taken  from  the  trachea,  the  cells  are 
surrounded  by  a  large  capsule;  this  capsule  appears  in  cultures  freshly 
taken  from  the  organs  attacked  and  in  very  old  cultures,  but  in  gen- 
eral does  not  exist  in  cultures. 

This  yeast  develops  easily  on  acid  substrate,  especially  on  agar 
and  beer  wort.  At  the  end  of  12  days  it  forms  white  colonies,  moist, 
round,  confluent  and  becoming  brown  in  old  cultures.  In  peptone 
broth  it  causes  a  cloudiness  and  later  a  delicate  precipitate  in  the 
bottom.  Still  later  it  causes  a  flocculent  deposit  at  the  bottom  of  the 
culture  flask.  On  gelatin  streaks  it  produces  a  white,  moist  growth; 
on  stabs  the  growth  is  established  along  the  line  of  inoculation  with 
fine,  round,  isolated  colonies.  On  the  surface  it  forms  a  sort  of  a 
button.  On  gelatin  plates  the  colonies  are  round  and  white.  On 
dextrose  agar  there  is  a  production  of  gas,  but  no  gas  is  formed  in 
manose,  maltose  or  saccharose  agar.  On  beer  wort  the  growth  con- 
sists of  a  dry  grayish-white  layer  of  round  confluent  colonies. 

This  yeast  is  very  pathogenic  for  rabbits. 

ATELOSACCHAROMYCES   OF  HUDELO.    De  Beurmann 
and  Gougerot 

This  yeast  was  found  by  Hudelo,  Duval  and  Loederich  l  in  a  human 
Saccharomycosis,  manifested  especially  by  a  periostitis  of  the  tibia. 
The  cells  are  refractive,  spherical  (2-20  JJL  in  diameter),  sometimes  oval 
or  elongated  into  short  sausage-shaped  cells.  No  filaments  are  formed. 

The  species  grows  easily  in  ordinary  media,  especially  on  carbo- 
hydrate media  with  slight  acid.  The  optimum  temperature  is  situated 
at  about  22°,  but  growth  is  accomplished  even  up  to  38°.  On  carbo- 
hydrate agar  white  streaks  are  formed  which  are  opaque  and  moist ;  on 
gelatin  there  is  meager  development  with  no  liquefaction;  on  potato 
whitish  streaks  are  formed,  later  becoming  ochre  colored,  and  finally 
a  reddish  black  pellicle  is  formed. 

This  yeast   inverts  saccharose,   but   does  not   decompose  lactose. 

1  Hudelo,  Duval  and  Loederich,  Un  cas  de  Blastomycose  a  foyers  multiples. 
Bull,  et  mem.  de  la  Soc.  de  med.  des  hop.  de  Paris,  1906. 


ATELOSACCHAROMYCES   HARTERI  359 

It  does  not  ferment  dextrose  or  maltose.  It  is  pathogenic  for  mice, 
less  pathogenic  for  rats,  guinea  pigs,  rabbits  and  dogs.  Intraperi- 
toneal  injection  causes  fatal  septicemia  in  mice. 

ATELOSACCHAROMYCES  OF  BREWER  AND  WOOD 

De  Beurmann  and  Gougerot 

This  yeast  was  isolated  by  Brewer  and  Wood  from  a  human 
blastomycosis.  In  situ  the  cells  are  spherical  (10-25  fjc  in  diameter) 
and  are  surrounded  by  a  large  mucilaginous  capsule.  In  culture  no 
filaments  are  formed,  but  the  cells  sometimes  remain  united  in  short 
chains  in  old  cultures.  On  glycerol  agar  the  growth  is  small  and  gives 
grayish  white  colonies.  On  agar  plates  development  is  very  abun- 
dant on  the  surface  in  a  creamy  yellow  mass.  On  potato  growth  is 
difficult.  On  gelatin  there  is  no  liquefaction.  The  species  grows 
difficultly  in  liquid  media  and  produces  no  fermentation. 

ATELOSACCHAROMYCES  HARTERI   (Harter2) 
De  Beurmann  and  Gougerot 

This  yeast  was  isolated  by  Harter  from  a  generalized  human 
saccharomycosis.  The  cells  are  oval  or  elliptical,  rather  spherical 
(4-6 /z  by  3-5  (JL).  On  solid  media  and  on  Raulin's  solution  sometimes 
elongated  units  are  observed,  but  never  filaments,  properly  speaking. 

On  old  cultures  on  carrot  certain  cells  become  round  and  very 
voluminous  (5-8 /^  in  diameter).  These  are  probably  durable  cells  or 
chlamydospores. 

The  yeast  grows  well  at  37°  at  laboratory  temperatures.  The 
growth  ceases,  however,  at  about  10°.  The  cells  withstand  55°,  but 
are  killed  in  a  quarter  of  an  hour  at  65°  in  moist  condition. 

On  gelatin  development  is  small  and  less  abundant,  white,  granu- 
lar, and  penetrating  into  the  medium  with  arborescent  structure. 
There  is  no  liquefaction  on  gelatin.  On  1%  glucose  gelatin  develop- 
ment is  a  little  more  abundant.  On  plain  agar  development  is  feeble 
and  slow.  On  glycerol  gelatin  there  is  abundant  growth  with  a  pro- 
duction of  a  downy  appearance  at  depths.  On  glucose  or  maltose 
agar  there  is  abundant  development  of  a  white,  creamy  growth.  On 
blood  serum  there  is  very  meager  growth.  On  carrot  there  is  abun- 
dant development,  quickly  covering  the  whole  surface  with  a  creamy, 
white,  thick,  granular  layer.  On  potato  growth  is  grayish  white,  dry 
and  not  very  abundant.  The  yeast  inverts  saccharose  very  slightly, 
but  does  not  give  any  fermentation. 

1  Brewer  and  Wood,  C.,  Blastomycosis  of  the  spine.    Annals  of  Surgery,  1908. 

2  Harter,   G.,   De  la  blastomycose  humaine.     These  de  medecine.     Nancy, 
1909. 


360  PATHOGENIC   YEASTS 

MERCIER'S1  YEAST 

This  yeast  was  found  by  Mercier  in  the  Blattes  (Periplaneta 
orientalis)  in  which  the  cells  existed  in  the  adipose  tissue  under  the 
form  of  round  units,  sometimes  oval,  with  a  very  finely  developed 
membrane.  The  parasite  grows  on  bouillon  and  gelatin  media.  The 
colonies  are  white.  The  optimum  temperature  for  budding  is  from 
22  to  25°  C.  • 

SACCHAROMYCES  CONOMELI  LIMBATI.     Karel  Sulc2 

This  yeast  has  been  found  by  Sulc  in  the  pseudovitellius  of  an 
Homoptera  Conomelus  limbatus.  The  cells  are  elliptical  or  oval, 
some  biscuit  shaped  having  an  alveolar  content  with  small  meta- 
chromatic  granules  in  the  vacuoles,  and  a  little  central  or  parietal 
nucleus.  The  buds  form  toward  the  end.  The  cells  are  often  united 
two  by  two. 

Sulc  has  also  found  Sack,  pseudococci  farinosi  which  lives  in  the 
pseudovitellius  on  another  Homoptera,  Pseudococcus  farinosus.  These 
yeasts  probably  live  symbiotically  with  the  insect. 

A  great  many  other  yeasts  have  been  described  in  different  in- 
fections of  men  and  animals;  for  instance,  Maggiora  and  Gradenigo 
have  isolated  in  a  case  of  otitis  S.  roseus;  Domingos  Freire  has  ob- 
served in  a  case  of  yellow  fever  the  presence  of  Cr.  xanthenicus; 
Flava  has  found  in  a  case  of  variola  the  Cr.  albus.  On  the  other  hand 
Goetano  has  isolated  the  Cr.  septicus  which  causes  a  rapid  fatal  sep- 
ticemia  in  guinea  pigs.  Castellani  has  described  in  various  tropical 
blastomyces  S.  cantliei,  Samboni,  and  Krusei,  also  Cr.  Lowi.  San 
Felice  has  isolated  S.  canis  I  and  II  which  provoke  tumors  in  a  dog. 
Finally,  Dangeard  has  pointed  out  in  the  bodies  of  Anguillules  the 
S.  anguillulae  which  causes  in  these  animals  a  very  deadly  malady. 
However,  the  morphological  and  biological  characters  of  these  yeasts 
have  not  been  described;  for  that  reason  there  will  be  no  description 
of  them  at  this  time. 

1  Mercier,  L.,  Un  organisme  a  forme  levure,  parasite  de  la  Blatte.    C.  R.  de 
la  Soc.  de  BioL,  v.  LX,  1906. 

2  Karel   Sulc,    Pseudovitellius   und   ahnliche    Gewebe   der   Homopteren   sind 
wohnstatten  symbiotischer  Saccharomyceten,  Sitzungsberichte  der  Konig.  Bohm. 
Gesellsch.  der  Wissenschaften  in  Prag,  March  30,  1910. 


CHAPTER  XIII 
FUNGI  RELATED  TO   THE  YEASTS 

IT  is  deemed  advisable  to  consider,  at  this  time,  a  few  of  the  fungi 
belonging  to  the  family   Endomyces  l  or  in  a  doubtful   position 
such  as  the  Monilia  and  Pseudomonttia;  these  are  set  apart  from 
the  yeast  by  the  greater  complexity  of  their  mycelium  but  the  physio- 
logical   and    certain    of    their   morphological    characteristics   resemble 
closely  the  Saccharomycetes  from  which,  at  times,  they  are  separated 
with  difficulty. 

ENDOMYCES  ALBICANS.    Vuillemin 

Syn.:  APOROTRICHUM    GRUBY  OIDIUM  ALBICANS.     Robin.  —  SYRINGO- 
SPORA  ROHINI.    Quinquaud.  —  SACCHAROMYCES  ALBICANS.    Reess. 

—  MONILIA  ALBICANS.      PlilUfc.  —  DEMATIUM  ALBICANS.      Laurent 

This  fungus,  which  causes  a  sickness  known  as  thrush,  has  been 
regarded  in  turn  as  an  (tidiwn,  a  Monilia  and  a  yeast.  Since  the  work 
of  Vuillemin  it  has  been  regarded  as  a  member  of  the  genus  Endo- 
myces. In  situ  and  in  cultures,  Endomyces  albicans  has  somewhat  the 
same  characteristics.  It  possesses  a  mycelium  with  cross  walls  and 
branches  more  or  less  well  developed;  yeast  structures  result  by  bud- 
ding from  the  branches.  (Fig.  155,  1  and  2.)  The  mycelium  never 
acquires  a  marked  differentiation  and  Endomyces  albicans,  speaking 
generally,  is  close  to  the  yeasts.  Both  structures,  yeast-like  and  myce- 
lium, are  able  to  change  one  into  the  other.  The  filaments  seem  able 
to  form  the  yeast-like  bodies  and  these  latter  the  filaments.  Myce- 
lium formations  are  more  or  less  well  developed,  depending  on  the 
conditions.  In  certain  cultures  the  yeast-like  structures  predominate 

1  The  genus  Endornyce*  is  characterized  by  a  typical  branched  mycelium 
with  cross  walls,  forming  yeast -like  bodies  or  oidia  and  chlamydospores,  and 
with  ascs  containing  4  ascospores.  These  are  formed  always  at  the  expense  of 
cells  in  the  mycelium,  most  often  at  the  end  of  a  branch,  exceptionally  in  some 
cell  in  the  mycelium.  In  certain  species,  the  formation  of  the  asc  is  preceded  by  a 
copulation  iso-  or  heterogamic.  The  genus  Endomyces  is  differentiated  from  the 
Saccharomyces  by  the  formation  of  a  typical  mycelium  and  the  formation  of 
ascs  in  mycelial  cells  and  never  in  the  yeast-like  structures.  However,  certain 
species  have  (End.  javanensis)  intermediate  characters  between  the  Endomyces 
and  Saccharomycetes  which  makes  it  difficult  to  class  them  with  either  one  of 
these  two  families. 

361 


362 


FUNGI  RELATED  TO  THE  YEASTS 


with  the  mycelium  -reduced  to  its  simplest  form.  In  other  media 
the  filaments  are  most  common.  Cultures  on  slices  of  carrot  have 
quite  a  development  of  mycelium  while  in  Raulin's  solution  growth  is 
almost  solely  of  the  yeast-like  structures.  According  to  Roux  and 
Linossier,1  the  yeast  structure  is  the  normal  one  while  the  mycelial 
form  appears  only  under  conditions  which  reduce  the  vitality.  Ac- 
cording to  Vuillemin,  on  the  contrary,  the  filamentous  form  is  the  one 
which  is  normal  and  the  yeast-like  form  appears  only  under  bad  condi- 
tions of  food  supply.  The  yeast-like  struc- 
tures are  spherical,  oval  or  elongated,  and  of 
variable  dimensions.  On  Raulin's  solution 
they  become  rather  large  and  appear  as  large 
spherical  cells  somewhat  resembling  those  of 
S.  cerevisiae  (Raj  at 2).  Guilliermond  has 
shown  that  the  units  of  the  filaments  contain 
ordinarily  a  single  nucleus,  rarely  more,  and 
the  yeasts  are  always  uni-nuclear.  This  has 
been  confirmed  by  H.  Penau.3 

Roux  and  Linossier,  and  later  Vuillemin, 
have  established  in  old  cultures  the  production 
of  very  resistant  forms  comparable  to  chlamy- 
dospores.  These,  which  have  received  the 
name  of  chr6nispores  or  chlamydospores,  de- 
velop at  the  end  of  certain  filaments  in  the 
form  of  distended  cells  filled  with  glycogen  and 
surrounded  with  a  thick  membrane  with  three 
superimposed  layers  (Fig.  152,  4).  Changed  to  different  media,  these 
chlamydospores  germinate  and  produce  yeasts  or  filaments. 

Vuillemin  has  described,  on  the  other  hand,  internal  globules, 
(Fig.  155,  3)  absolutely  analogous  in  appearance  to  yeasts,  which  form 
on  the  interior  of  the  filaments.  The  author  considers  them  as  re- 
sistant forms. 

In  our  opinion,  these  internal  bodies  may  be  similar  to  those  which 
are  commonly  found  among  the  fungi.  There  are,  here  and  there,  the 
formation  of  yeasts  or  conidial  forms,  in  the  interior  of  an  inter- 
calary unit,  with  degenerating  contents,  by  the  budding  of  a  contiguous 
unit.  This  latter  buds  in  the  interior  of  a  dead  unit  which  is  near 

1  Roux,  G.,  and  Linossier,  G.    Rech.  morph.  sur  le  champ,  du  Muguet.     Arch, 
de  Med.  experim.  1890. 

2  Rajat,  H.,  Le  champ,  du  Muguet.     These  de  doct.  en  medecine,  Lyon,  1906. 

3  Penau,  H.,  Cytologie  de  L'End.  albicans  forme  levure,  v.  CLII,   1900,  and 
Cytologie  de  TEnd.  albicans  forme  mycelienne.  C.  R.  Ac.  des  Sciences,  v.  CLII, 
1910. 


Fig.  155.  —  Yeast  Forma 
from  a  Case  of  Thrush. 

2,  Mycelial  Forms  from  a  Case 
of  Thrush;  3,  Internal  Glob- 
ules; 4,  Chronispores;  5,  Asca 
(after  Vuillemin). 


ENDOMYCES   ALBICANS  363 

it;  the  cells  which  are  derived  from  the  budding  live  parasitically 
on  the  protoplasm  about  it  until  their  growth  breaks  it.  Analogous 
formations  are  frequent  among  the  Endomycetes.  Rose  has  described 
them  in  Endomyces  magnusii. 

The  ascs  of  E.  albicans  were  accidentally  discovered  by  Vuillemin 
on  old  cultures  on  beets,  without  which  this  author  would  have  been 
unable  to  determine  the  conditions  for  their  formation.  The  ascs  appear 
as  large,  oval  or  elliptical  cells,  4-5  ju  in  diameter,  formed  by  lateral 
budding,  or  at  the  terminal  of  the  units  of  the  mycelia,  or  sometimes 
derived  by  germination  of  the  chlamydospore.  They  possess  mem- 
brane enclosing  four  flattened  ascospores,  slightly  kidney  shaped, 
with  thick  walls  (Fig.  155,  5).  The  germination  has  not  been 
observed.  The  presence  of  these  ascospores  has  allowed  Vuillemin  to 
classify  the  fungus  for  thrush  in  the  genus  Endomyces. 

These  ascs  have  only  been  observed  by  Vuillemin  and  Daiereuva.1 
All  the  authors  who  have  searched  since  to  obtain  them,  have  failed  in 
their  attempts.  Also  certain  authors  have  thought  that  there  might 
exist  many  varieties  of  E.  albicans,  some  of  which  have  preserved 
their  sporogenic  properties  (Guegen,  Raj  at). 

This  opinion  seems  to  be  still  further  confirmed.  Raj  at  has  iso- 
lated three  varieties  of  the  fungus  of  thrush.  One  of  these  corre- 
sponds by  its  morphological  and  chemical  characteristics  to  that  species 
described  by  Vuillemin  although  it  has  not  shown  the  formation  of 
ascs.  The  other  two  types  present  morphological  characteristics 
very  different  from  the  type  species. 

Beauverie  and  Lesieur  have  isolated  from  the  blood  of  a  fatal 
septicemia  a  variety  of  Endomyces  albicans.  This  is  distinguished 
from  the  type  species  by  the  fact  that  it  ferments  lactose  and  exhibits 
different  cultural  characteristics  on  carrot.  Castelanni2  has  more 
recently  shown  the  plurality  of  the  thrush  fungus.  He  has  isolated 

29  different  fungi  from  thrush  cases.    He  also  separated  a  number  of 
new  races  of  the  thrush  fungus. 

All  of  these  fungi  belong  to  the  genus  Monilia  and  may  be  dif- 
ferentiated by  their  biochemical  characteristics.  Guilliermond  iso- 
lated three  types  of  the  thrush  fungus  from  infections  at  hospital 
No.  101  during  the  war,  at  Lyon.  Two  of  these  belonged  to  the  genus 
Monilia  and  the  third  was  a  typical  saccharomyces,  with  ascs  but 
not  corresponding  to  Saccharomyces  anginae  of  Troisier  and  Alchalme. 

1  Daiereuva,  M.,  Rech.  sur  le  champ,  du  Muguet.    These  de  me"decine  Nancy, 
1899. 

2  Castelanni,    A.     The   plurality   of  species  of  the   so-called  thrush  fungus 
(Champignon  du  muguet)  of  temperate  climates.     Annals  de  1' Institute  Pasteur 

30  (1916),  149. 


384  FUNGI   RELATED  TO   THE   YEASTS 

E.  albicans  develops  between  20  and  39°  C.  and  grows  on  solid  or 
liquid  media  slightly  acid;  no  scum  is  produced  on  the  surface  of 
liquid  media.  On  carbohydrate  liquid  media  and  fruit  juices  it  gives 
a  slight  growth  with  a  flocculent  sediment.  On  gelatin  plates  the  colo- 
nies are  round,  white  and  creamy,  and  it  produces  liquefaction  of  the 
gelatin.  In  gelatin  stabs  development  is  slight  and  superficial.  On 
agar  the  fungus  produces  a  white  line  which  thickens  to  a  creamy  layer 
at  first  thick  then  honeycombed.  On  potato  it  gives  small  colonies 
of  a  dirty  white  color  and  on  carrot  creamy  white  and  folded  growth. 
It  grows  with  difficulty  in  milk,  which  it  coagulates  in  20  to  30  days. 

E_.  albicans  causes  a  slight  fermentation  of  dextrose. 

Anderson  has  mentioned  the  very  frequent  presence  in  the  human 
intestines  of  a  fungus  very  closely  related  to  Endomyces  albicans 
to  which  he  has  given  the  name  of  Parasaccharomyces  Ashfordii. 

PARASACCHAROMYCES  ASHFORDIL    Anderson1 

"Morphology.  In  young  cultures  cells  are  round  or  slightly  oval; 
in  old  cultures  cells  are  of  many  forms:  oval,  elongated,  elliptical, 
round,  or  irregular;  giant  cells  are  common.  Septate  mycelium  de- 
velops in  gelatin  hanging-drop  and  in  old  cultures.  Budding  occurs 
from  any  point  on  the  young  cells,  but  usually  near  the  ends  of  articles 
in  old  cultures.  The  size  is  4.5  x  5  /z. 

"  Cultural  Characters.  On  glucose  agar  the  streak  is  filiform,  raised, 
glistening,  chalk^white  and  smooth;  later  the  central  portion  may  be- 
come rugose  or  pitted;  the  edge  of  the  streak  may  remain  entire  or 
may  become  decidedly  filamentous,  due  to  the  outward  growing  hyphal 
elements  under  the  surface  of  the  medium.  There  is  a  growth  in  gelatin 
stab  at  first  filiform,  later  it  develops  scattered,  bushy  clusters  of  fila- 
ments. In  liquid  sugar  mediums  and  beer  wort  a  very  evident  ring 
formation  occurs;  no  pellicle  is  present. 

(l Physiologic  Properties.  It  ferments  glucose,  maltose  and  levulose; 
occasionally  sucrose  and  galactose  are  fermented.  Yeast-water  sugar 
mediums,  with  an  initial  acidity  of  +  1,  become  more  alkaline.  Litmus 
milk  is  rendered  alkaline  in  2  weeks,  but  is  not  clotted.  Gelatin  is 
rarely  liquefied. 

"The  culture  was  isolated  from  a  sprue  patient  by  Dr.  B.  K. 
Ashford  in  Porto  Rico. 

"  This  species  strongly  resembles  the  fungus  variously  called  Oidium 
albicans,  Monilia  albicans,  and  Endomyces  albicans.  Castellani  ('16) 
has,  however,  reserved  the  name  Monilia  albicans  for  a  species  which 

1  Anderson,  H.  W.  Yeast-like  fungi  of  the  human  intestinal  tract.  Jour. 
Infectious  Diseases  21  (1917)  341-386. 


PARASACCHAROMYCES  THOMASII 


365 


alv/ays  clots  milk  and  liquefies  gelatin.  Monilia  albicans,  Oidium 
albicans  and  Endomyces  albicans  are  synonyms,  and  if  Vuillimin's  ('99) 
results  are  accepted  and  are  of  general  application  to  all  of  these,  the 
correct  name  for  the  species  is  Endomyces  albicans,  since  he  states 
that  this  species  forms  asci  after  the  manner  of  other  species  of  the 
genus  Endomyces.  Since  all  efforts  to  develop  the  perfect  stage  of 
the  sprue  organism,  both  by  Dr.  Ashford  and  myself,  ended  in  failure 
and  since  it  differs  in  many  of  its  physiologic  characters  from  the  typical 
Endomyces  albicans,  it  has  been  thought  best  to  give  it  specific  rank 
rather  than  to  regard  it  as  a  variety  of  Endomyces  albicans.'' 


PARASACCHAROMYCES  THOMASII.     Anderson1 

"Morphology.  In  young  cultures,  cells  are  elliptical  or  ovate; 
in  old  cultures,  surface  cells  are  round,  oval,  elliptical,  or  elongated; 
submedial  cells  form  a  distinct  mycelium  mostly  by  elongation  of  cells 
produced  by  budding.  There  is  occasional  septation  in  gelatin  hang- 
ing-drop. Budding  occurs 
from  ends  or  shoulders. 
The  size  is  3.5  x  5  p. 

11  Cultural  Characters. 
On  glucose  agar  the  streak 
is,  at  first,  white,  glistening, 
convex,  and  smooth;  later 
the  surface  becomes  rugose 
with  a  decidedly  elevated  Fig.  155-B.  —  Parasaccharomyces  Thomasii,  An- 
ridge  down  the  center.  derson. 

Beneath  the  surface  of  the, 
medium  the  radiating  hy- 
phae  form  a  villous  fringe.  In  beer  wort  and  liquid  sugar  mediums 
no  pellicle  or  ring  is  present.  In  gelatin-stab  cultures  the  growth  is 
finely  villous.  Giant  colonies  in  beer  wort  gelatin  are  decidedly  yellow 
in  color  and  otherwise  very  characteristic. 

"Physiologic  Properties.  Slow  fermentation  of  glucose,  levulose 
and  maltose.  In  litmus  milk  there  is  a  decided  alkaline  reaction. 

"  The  culture  was  isolated  from  human  feces. 

"  The  species  is  similar  to  Parasaccharomyces  Ashfordii  in  its  physi- 
ologic properties.  It  differs  mainly  in  its  morphologic  characters 
and  the  type  of  giant  colonies  produced.  The  yellow,  rugose  colony 
in  beer  wort  gelatin  is  especially  characteristic  and  easily  distinguishes 
in  this  species  from  P.  Ashfordii." 


1,  Cells  from  Young  Beer  Wort  Culture;  a,  Elongated  Cells 
Forming  a  Pseudo-mycelium  Beneath  the  Surface  of  an 
Agar  Slant;  b,  Cells  from  the  Surface  of  the  Same  Culture. 


1  Anderson,  H.  W.     Yeast-like  fungi    of  the  human  intestinal  tract. 
Infectious  Diseases  21  (1917)  341-386. 


Jour. 


366 


FUNGI   RELATED  TO   THE  YEASTS 


ENDOMYCES  LINDNERI.     Saito 

This  yeast  was  isolated  from  Chinese  yeast  by  Saito.  This  Chinese 
yeast  was  used  in  the  preparation  of  beer.  It  has  the  same  morpho- 
logical characteristics  as  Endomyces  jibuliger;  the  ascs  are  often  formed, 
as  in  Endomyces  liger,  from  an  anastomosis  taking  place  between  two 
cells  in  the  mycelium.  Maugenot  has  shown  that  these  anastomoses 
are  analogous  to  those  which  have  been  described  for  Endomyces 
fibuliger  and  never  result  from  a  copulation.  They  represent  what  is 
left  of  an  ancestral  sexuality.  Endomyces  Lindneri  is  very  closely 
related  to  Endomyces  fibuliger  and  is  distinguished  from  this  genus 
only  by  the  fact  that  it  ferments  maltose  and  dextrose  on  which 
Endomyces  fibuliger  has  no  action. 


ENDOMYCES  HORDEI.     Saito 

Saito  has  isolated  an  organism  which  possesses  certain  character- 
istics of  a  Monilia  with  fragments  of  my- 
celium with  cross  walls  forming  yeast-like 
structures  as  conidia.  When  inoculated 
into  media,  these  germinate  into  a  budding 
mycelium.  In  the  sediment,  they  develop 
especially  as  yeasts.  The  mycelium  is  also 
able  to  break  up  into  fragments  like  oidia 
in  old  cultures.  The  ascs  appear  in  old  cul- 
tures on  agar  and  gelatin.  On  plate  cul- 
tures, they  form  in  great  numbers  at  the 
end  of  three  days.  They  are  formed  by 
budding  of  the  units  of  the  mycelium  under 
the  form  of  large  round  cells  (6-12  /z).  The 
ascospores  are  to  the  number  of  from  2  to 
4  per  asc,  and  are  hat  shaped  (3-4 /A).. 
They  are  provided  with  an  exosporium 
and  an  endosporium.  During  germination 
the  exosporium  breaks  and  the  ascospore 
germinates  by  ordinary  budding. 

The    growth    on    plates    is    under    the 
form  of  small  moist  patches  with  filaments. 
On  beer  wort  or  decoction  of  "Koji"  the 
3    fungus  forms  a  very  thick  scum  which  is 

^^    ^    ^    ^    ^^    ^    &    ^^    ^^ 

mental  growth. 
The  optimum  temperature  for  growth  is  30°  C.     The  yeast  will 


Fig.  155-C.  —  Endomyces 
H  or dei. 

1,  Budding  Mycelium;     2,  Ascs; 
Germination  of  Ascospores   (after 
Saito). 


ENDOMYCES  CAPSULARIS  367 

vegetate,  however,  between  15°  and  40°  C.  It  induces  a  fermentation 
in  beer  wort  very  slowly  and  gives  off  an  ethereal  odor.  It  acts  en- 
ergetically on  saccharose,  maltose,  dextrose,  levulose,  mannose,  galac- 
tose,  raffinose,  dextrine,  xylose  and  arabinose  and  feebly  on  rhamnose 
and  a-methylglucoside. 

Endomyces  Hordei  resembles  Endomyces  fibuliger  very  closely.  It 
is  also  related  to  Endomyces  Lindneri.  The  formation  of  the  asc  by 
simple  budding,  without  any  trace  of  sexuality,  is  responsible  for  this 
resemblance. 

ENDOMYCES   CAPSULARIS.     Guilliermond 

Syn.:  SACCHAROMYCES  CAPSULARIS.     Schionning l 

This  yeast  was  described  in  1903  by  Klocker  who  isolated  it  from 
pastureland  in  the  Swiss  Alps.  Guilliermond  finally  subjected  it  to 
careful  study. 

Endomyces  capsularis  for  the  most  part  has  cells  in  the  mycelial 
and  the  yeast  form  at  the  same  time.  It  vegetates  at  the  bot- 
tom of  the  medium  as  a  sediment  or  in  the  form  of  a  scum.  The 
mycelium  is  especially  well  developed  in  the 
scums  or  on  solid  media.  It  is  branched, 
with  cross  walls,  and  according  to  the  in- 
vestigations of  Guilliermond,  always  has  a 
single  nucleus.  It  is  able  to  present  dif- 
ferent appearances.  '  Some  of  the  filaments 
remain  sterile  while  others  form  by  lateral 
and  terminal  budding  numerous  yeast-like 
cells.  Rarely  there  are  others  which  form 
small  septa,  dividing  the  thread  into  units 
which  break  off  like  oidia. 

The  yeast-like  bodies  develop  especially 
in  growths  of  sediment.  They  look  like 
true  Saccharomyces;  their  form  is  ellipsoidal  spores  (after  Schi°nning)- 
or  oval  like  Saccharomyces  Pastorianus  or  Saccharomyces  ellipsoideus 
(Fig.  156  d).  Many  among  them  have  a  point  at  one  or  both  ends. 
They  never  have  but  a  single  nucleus.  Aside  from  the  yeast-like  struc- 
tures, one  may  find  some  elongated  or  walled  cells  which  represent 
yeasts  in  the  process  of  making  up  a  mycelium. 

The  optimum  temperature  for  vegetation  is  situated  between 
25  and  28°  C.  The  maximum  temperature  is  38.5°  C.  and  the  mini- 
mum about  0.5°  C.  The  ascs  appear  under  the  same  conditions  which 

1  Schionning,  H.  Nouveau  genre  de  la  famille  des  Sacch.  Comp.  Rend,  du 
lab.  de  Carlsberg.  Vol.  6,  Book  II,  1904. 


368 


FUNGI   RELATED   TO   THE  YEASTS 


determine  the  sporulation  of  yeast  (plaster  blocks,  cultures  in  yeast 
water  and  slices  of  carrot).     The  optimum  temperature  for  sporula- 
tion on  plaster  blocks  is  between  25°  C.  and  28°  C.,  the  maximum  is 
34.5°-35°  C.  and  the  minimum  5-8°  C.     The  ascs  always  form  at  the 
expense  of  units  in  the  mycelium  and  never  of  the  yeast-like  struc- 
tures.    Finally,  they  only  appear  in   contact 
with  air  on  solid  substrates  and  in  scums.     The 
ends  of  the  threads   separate  into  cells  which 
become  round  and  produce  spherical  or  elon- 
gated  forms    similar    to    oidia.     These    cells 
develop   either  by  constriction   or  transverse 
partition  or  by  a  process  intermediate  between 
Fig.  157.  —  Ascospores  in  these  two  processes.     The  cells  thus  -formed 
Endomyces  capsularis  (ac-  sweu  up  an(j  snow  a  granular  contents  very 
cording  to  Schionnmg).         r      x-         i  A         u        •  j      n      •   ^ 

refractive,  later  changing  gradually  into  ascs 

(Fig.  58,  2  and  3,  and  Fig.  157,  a).  Often  the  ases  are  able  to  form 
from  an  intercalary  cell  in  the  mycelium  which  enlarges  and  becomes 
round  (Fig.  157,  b).  The  investigations  of  Guilliermond l  indicate 
that  the  ascs  possess  a  single  nucleus  like  those  of  the  Saccharomycetes. 
A  karyogamy  does  not  take  place  here  as  with  Exoascus.  The  ascs 
almost  constantly  possess  4  ascospores. 

The  ascospores  are  very  resistant  to  acids.  If  the  mycelium  which 
has  produced  ascs  is  treated  with  a  strong  solution  of  sulfuric  acid 
or  other  mineral  acids,  the  mycelium  and  the 
ascs  dissolve.  On  the  other  hand,  the  asco- 
spores resist  and  take  on  a  beautiful  red  color. 
The  ascospores  of  the  Saccharomycetes  are, 
on  the  other  hand,  strongly  attacked  by  these 
acids  and  not  colored  a  rose  color.  The  as- 
cospores are  ellipsoidal  or  oval  (3.5  to  8  JJL  in 
diameter).  They  possess  a  double  membrane, 
an  exosporium  and  an  endosporium.  The  Fi 
exosporium  is  formed  of  two  valves  in  which 
the  adjacent  edges  cause  a  sort  of  projecting 

ring  by  means  of  which  the  ascospores  resemble  those  of  Willia 
Saturnus.  This  ring  separates  the  ascospore  into  two  unequal  parts 
(Fig.  158). 

Reaching  the  adult  stage,  the  ascospores  absorb  their  wall  quite 
rapidly,  setting  free  the  ascospores,  but  these  more  often  remain  united 
in  groups  of  four.  When  the  ascospore  germinates,  the  exosporium 
cracks  to  form  two  unequal  parts  which  remain  united  at  the  point 

1  Guilliermond,  A.  Recherches  cytologiques  et  taxonomiques  sur  les  Endomy- 
cetees.  Rev.  gen.  Bot.  21,  1909. 


158.  —  Germination  of 
scospores  in  Endomyces 
cavsularis. 


ENDOMYCES  CAPSULARIS  369 

for  some  time.  Germination  of  the  ascospores  is  accomplished  either  by 
the  formation  of  a  germinating  tube  or  by  budding.  The  germinating 
tube  becomes  the  point  of  beginning  of  the  mycelium.  (Fig.  156  e,  and 
158.)  On  beer  wort  the  ascospores  germinate  by  budding,  only  the 
yeast-like  cells,  which  elongate  without  separating,  show  a  tendency  to 
form  a  mycelium,  but  never  a  true  mycelium.  In  yeast  water  and  on 
slices  of  carrot,  on  the  contrary,  ascospores  are  never  formed  from 
budding  but  a  filament  forms  walls,  thus  forming  directly  a  mycelium. 

In  certain  unfavorable  conditions  for  growth  of  this  fungus,  the 
ascospores  may  form  a  bud  or  a  germinating  tube  which  changes 
directly  into  an  asc.1 

Endomyces  capsularis  develops  for  the  most  part  in  artificial 
media.  On  beer  wort  at  25°  C.,  after  about  one  day,  it  forms  on  the 
surface  a  deposit  of  yeast  which  sets  up  the  alcoholic  fermentation. 
After  two  days,  there  are  formed  on  the  surface  of  the  wort  small 
floating  patches  of  scum  made  up  of  a  typical  mycelium.  After  a 
prolonged  repose  the  scum  finally  covers  the  whole  surface;  this  scum 
is  frequently  situated  above  great  bubbles  of  froth  which  make  it 
uneven.  If  the  culture  is  left  in  quiet  repose,  the  scum  forms  a 
thick  cover  on  the  surface,  very  uneven,  dry  and  white  and  slightly 
velvety,  composed  of  a  mixture  of  yeast-like  structures  and  mycelium. 

In  yeast  water,  this  fungus  forms  on  the  surface  of  the  medium 
at  the  end  of  two  days  white  islands  of  scum  composed  of  a  mycelium 
in  which  some  of  the  mycelial  threads  form  yeast  bodies  by  con- 
striction. After  foiy  days,  the  entire  surface  is  covered  with  quite  a 
thick  mycelium,  slightly  velvety  in  appearance.  Vegetation  is  then 
formed  of  a  typical  mycelium  well  developed,  in  which  the  ends  pro- 
duce many  ascs.  On  must  gelatin,  a  dry  velvety  growth  is  produced 

1  According  to  Klocker  and  Dombrowski,  the  Saccharomycetes  are  distinguished 
from  the  Endomycetes  by  the  fact  that  in  the  first  the  ascospores  are  able,  under 
certain  conditions,  to  change  directly  into  a  new  asc  without  preliminary  multiplica- 
tion while  in  the  second,  the  ascs  only  form  after  the  formation  of  a  mycelium. 
These  authors  think  that  this  peculiarity  makes  a  better  differential  characteristic 
between  the  Endomycetes  and  the  Saccharomycetes.  According  to  Klocker,  En- 
domyces capsularis  may  be  classed  among  the  Saccharomycetes  because  in  this 
fungus  the  ascospores  are  capable  of  producing  ascs  in  their  germination.  On 
the  contrary,  Endomyces  fibuliger  may  be  classed  among  the  Endomycetes  be- 
cause the  ascospores  never  change  into  ascs.  Klocker  and  Dombrowski  have  only 
observed  these  two  endomyces  and  have  not  investigated  whether  the  spores  of 
other  members  might  not  change  into  ascs.  Indeed,  one  ought  not  to  attribute 
too  much  importance  to  the  opinions  of  these  two  investigators,  who  base  their 
statements  on  too  hasty  generalizations.  What  connects  Endomyces  capsularis  to 
the  Endomycetes  is  the  high  differentiation  of  its  mycelium  and  the  mode  of  forma- 
tion of  its  ascs  always  at  the  expense  of  the  mycelium.  This  fungus  is,  however, 
so  closely  related  to  Endomyces  fibuliger  that  a  separation  is  merely  arbitrary. 


370  FUNGI   RELATED  TO  THE   YEASTS 

with  a  grayish  white  color.  The  gelatin  is  not  liquefied.  On  must 
agar,  the  growth  is  dry,  much  folded  and  velvety,  assuming  a  chocolate 
color  after  a  time.  This  fungus  ferments  maltose,  dextrose,  levuloso, 
and  d-galactose,  but  does  not  act  on  1-arabinose,  ramnose,  lactose 
and  saccharose.  It  does  not  secrete  invertase. 

Schionning  has  described  this  fungus  as  a  Saccharomycete  which 
he  classes  along  with  Saccharornycetes  guttulikus  in  the  genus  Saccharo- 
mycopsis  characterized  with  its  ascospores  in  a  double  membrane. 
On  account  of  the  high  differentiation  of  its  mycelium  and  the  for- 
mation of  ascospores  almost  always  at  the  end  of  the  filaments  of  th;? 
mycelium  and  never  at  the  expense  of  yeast -like  cells,  we  have  been 
led  on  the  contrary  to  class  it  with  Endomyces  fibuliger  and  put  it 
into  the  genus  Endomyces  under  the  name  of  End.  capsularis.  It 
will  be  demonstrated  in  the  following  paragraph  that  this  fungus  is 
very  closely  related  to  Endomyces  fibuliger. 

ENDOMYCES  FIBULIGER.     Lindner1 

Endomyces  fibuliger  was  discovered  in  1908  by  Lindner  on  bread 
where  it  formed  white  spots  resembling  chalk  and  caused  a  trouble 
known  as  "  chalky  bread."  By  the  investigations  of  Lindner,  Dom- 
browski2  and  Guilliermond,3  this  yeast  is  well  known  to-day.  In 
cultures  it  has  a  typical  mycelium  with  cross  walls  and 
branches  in  each  unit  of  which  there  is  a  nucleus.  The 
filaments  of  this  mycelium  at  times  form  conidia,  yeast- 
like  structures  and  ascs. 

The  conidia  appear  only  in   that  part  of  the  my- 
celium that  is  directly  exposed  to  the  air,  that  is,  in 
the  scum  on  the  surface  liquid  media  or  in  the  upper 
reaches  of  the  growth  on  solid  media.     They  are  formed 
Formation^   m  §rea^  abundance  under  these  conditions  and  show  a 
Conidia     in    white  powdery  appearance.     These  conidia  either  form 
G  ?    directly  from  the  mycelium  by  budding,  or  form  at  the 
expense  of  budding  cells  like  the  yeasts  which  are  formed 
by  branches  in  the  mycelium.     (Fig.  159.)    They  separate  from  the 
units  which  form  them  and  leave  a  sort  of  sterigmata  which  remains 
attached  to  the  latter  cells.    The  conidia  look  like  grape  seeds  which 
are   provided  with  a  thick  membrane,  a    protoplasm  filled  with  fat 

1  Lindner,  P.     Endomyces  fibuliger  n.  sp.    Wochensch.  Brau.  No.  24.  1908. 

2  Dombrowski,   W.     Sur  1'End.   fibuliger.     Comp.  Rend.   lab.   de   Carlsberg, 
Vol.  7,  Book  4,  1909. 

3  Guilliermond,    A.      Recherches   cytologiques   et   taxonomiques   sur   les   En- 
domycetees.    Rev.  gen.  Bot.  21,  1909;   Remarques  sur  le  develop.  1'End.  fibuliger. 
Comp.  Rend.  Soc.  Biol.  67,  1910. 


ENDOMYCES  FIBULIGER  371 

globules  and  a  single  nucleus.  The  outer  part  of  the  membrane  is 
easily  detached  when  the  conidia  separate  from  the  cells  which  form 
them.  The  conidia  have  a  tendency  to  reunite  in  small  masses  sur- 
rounded by  bubbles  of  air.  A  sort  of  network,  mucilaginous  in 
character,  is  formed  which  is  quite  comparable  to  that  formed  by  the 
Saccharomycetes.  This  network  is  destroyed  by  heat.  Possibly  it 
constitutes  a  means  of  preservation  for  the  conidia. 

The  conidia  never  bud  in  the  media  in  which  they  are  formed. 
Only  when  transferred  into  a  fresh  medium  is  it  that  they  bud  either 
by  yeast-like  structures  or  by  sending  out  a  germinating  tube  which 
branches  to  form  a  mycelium. 
They  represent,  then,  forms 
which  are  comparable  to  the 
Chlamydospores  of  other  Endo- 
mycetes. 

In  parts  of  the  mycelium 
which  are  situated  in  scantily 
aerated  locations,  as  in  the  sedi- 
ment in  a  liquid  culture  or  deep 
down  into  a  solid  medium,  the 

mycelium  never  produces  conidia 

,  , ,  £  Fig.    160.  —  Germination  of  Ascospores  in 

but,    on   the    contrary,  forms  a  Endomyces fibuliger. 

large  number  of  yeast-like  struc- 
tures. (Fig.  55.)  These  vary  in  their  shapes  and  sizes.  In  -certain 
media,  they  are  smaller  than  conidia  and  resemble  the  Mycoderma. 
Sometimes,  they  may  be  much  larger  than  the  conidia  and  possess  a 
round  shape.  They  contain  but  a  single  nucleus.  These  yeasts,  after 
being  detached  from  the  mycelium  for  a  time,  continue  to  bud  in  the 
medium  in  which  they  are  formed  and  furnish  new  generations  of  the 
yeasts.  On  fresh  media,  they  elongate  and  furnish  a  mycelium  or 
germinate  into  yeast-like  structures. 

Often  there  may  be  seen  in  parts  of  the  mycelium  that  form  these 
yeast-like  bodies,  a  sort  of  dissociation  of  filaments.  The  walls  come 
nearer  and  the  units  which  are  thus  formed  separate  into  elongated 
cells  which  look  like  oidia. 

Lindner  has  noticed,  in  certain  cases,  the  formation  in  the  inter- 
calary units  of  the  filaments,  of  internal  cells  which  he  ignores,  but 
which  seem  to  us  to  be  conidia  or  yeast  structures. 

The  ascs  are  formed  under  the  same  conditions  as  with  the  Sac- 
char omyces.  They  appear  quickly  if  a  piece  of  the  mycelium,  young 
and  well  nourished,  is  placed  in  a  covered  dish  containing  a  thin  layer  of 
distilled  water  or  on  different  solid  media  (slices  of  carrot)  as  well  as  in 
most  old  cultures.  The  most  favorable  temperature  for  sporulation 


372  FUNGI   RELATED   TO  THE   YEASTS 

is  situated  at  about  20°  C.  At  this  temperature  the  ascs  appear 
in  about  72  hours.  Like  the  conidia,  the  ascs  are  always  formed  in  the 
presence  of  air.  They  are  able  to  appear  in  the  same  time  and  numer- 
ous filaments  may  be  found  which  form  both  ascs  and  conidia.  They 
form,  as  with  Endomyces  capsularis,  at  the  ends  of  the  filaments  either 
by  budding,  or  by  partition,  followed  by  a  separation  of  terminal  units 
which  gives  a  chain  of  ascs.  Sometimes  they  are  formed  by  an  inter- 
calary unit. 

The  formation  of  these  ascs  is  of  special  interest  because  of  the 
anastomosis  which  takes  place.  In  most  cases  the  ascs  are  formed 
without  anastomosis,  as  in  the  two  preceding  species  of  Endomyces, 
but  in  about  half  of  the  cases,  the  young  bud  destined  to  form  an  asc 
anastomoses  with  a  cell  situated  in  the  vicinity  by  means  of  a  sort  of 
copulation  canal.  This  phenomenon  has  been  sufficiently  described  in 
a  preceding  chapter.  Let  us  recall,  however,  that  the  middle  wall 
that  separates  the  asc  from  the  cell  with  which  it  is  anastomosing  does 
not  disappear  but  persists  in  the  copulation  canal.  Sometimes  this 
wall  does  disappear  but  even  in  this  case  there  is  produced  no  mix- 
ture of  the  contents  of  the  two  cells.  (Figs.  56  and  57.)  Also  we 
should  look  on  these  anastomoses  as  traces  of  an  ancestral  reproduction 
analogous  to  that  of  Eremascus  fertilis,  but  having  disappeared  today. 

The  ascs  appear  like  large  round  or  oval  cells.  They  have  but  one 
nucleus  and  do  not  possess  karyogamy.  They  contain  numerous  asco- 
spores  which  may  vary  from  one  to  four,  but  usually  four.  The 
ascospores  are  hemispherical  and  they  are  surrounded  by  a  projecting 
ring  which  makes  them  look  like  a  hat.  In  this  manner  they  are  like 
the  ascospores  of  Willia  anomala  and  Endomyces  decipiens. 

Germination  of  the  ascospores  has  been  recently  studied  by  Dom- 
browski.  The  ascospores  are  provided  with  a  double  membrane,  an 
exosporium  and  an  endosporium.  At  the  moment  of  germination, 
the  ascospores  take  their  usual  form  if  in  a  young  culture  or  become 
globular  if  in  an  old  culture;  the  exosporium  opens  up  at  any  place 
on  the  surface  of  the  ascospore.  These  germinate  indifferently  either 
to  produce  yeast-like  bodies  or  to  form  a  mycelium  directly.  (Fig.  160). 

The  ascospores  are  incapable,  as  with  Endomyces  capsulatus,  of 
yielding  ascs  directly.  The  ascs  are  only  produced  when  the  mycelium 
is  well  developed.  Endomyces  fibuliger  develops  quickly  on  most 
media.  In  beer  wort,  after  three  weeks,  it  shows  a  dry  scum  formed  of 
mycelium,  covered  by  numerous  conidia  which  give  it  a  farinaceous 
appearance;  in  the  sediment,  yeast-like  cells  are  formed.  In  course 
of  development,  an  agreeable  aroma  is  given  off  with  a  feeble  fer- 
mentation. 

In  wort  gelatin,  it  develops  with  a  dry  farinaceous  spot  and  brings 


ENDOMYCES  JAVANENSIS 


373 


about  a  liquefaction  of  the  gelatin  after  a  week.  The  vegetation 
is  made  up  of  a  thick  mycelium  which  gives  numerous  conidia.  In 
wort  agar,  the  colonies  are  of  a  chocolate  color.  On  carrot,  this  fungus 
develops  abundantly  with  a  mycelium  which  in  the  beginning  furnishes 
many  conidia;  later  after  18  days,  there  is  an  abundant  production 
of  ascs.  The  deep  portions  of  the  mycelium  form  many  yeasts. 

Endomyces  fibuliger  ferments  saccharose  actively  and  less  actively 
dextrose,  d-mannose  and  levulose;  it  ferments  feebly  raffinose,  lac- 
tose, d-galactose  and  a-methylglucosides.  It  has  no  action  on  maltose, 
dextrine,  arabinose,  xylose,  trehalose,  melibiose,  mannite  and  inuline. 
Endomyces  fibuliger  is,  in  general,  closely  related  to  Endomyces  capsu- 
laris.  It  resembles  it  by  the  complexity  of  its  mycelium^  its  yeast 
structures  and  the  mode  of  formation  of  ascs  and  is  distinguished  only 
by  the  formation  of  conidia  and  the  traces  of  an  ancestral  copulation 
which  it  has  kept. 


ENDOMYCES   JAVANENSIS.     Klocker 

This  species  was  discovered  in  1909  by  Klocker  l  in  soil  from  Java. 
The  vegetation  is  composed  in  part  of  cells 
like  yeasts  and  in  part  of  a  mycelium  with 
walls.  The  mycelium  offers  a  slight  tendency 
to  separate  its  units  like  oidia.  However,  it 
is  much  more  reduced  than  in  Endomyces 
capsularis  and  Endomyces  fibuliger  (Fig. 
161).  The  yeast  cells  (7  to  9 /*  in  length) 
often  look  like  lemons  but  some  look  like  a 
spindle;  others  are  ellipsoidal,  spherical,  in 
the  form  of  a  sausage  or  very  much  elon- 
gated. In  a  general  manner,  they  resemble 
the  cells  of  Saccharomyces  apiculatus  very 

much.  The  temperature  limits  for  growth  Fig"  m  _Endomyce8  Java. 
are:  maximum  36°-38°  C.,  minimum,  5°  to  nen^is._  Mycelium  with 
10°  C. 

Sporulation  is  abundant  in  liquid  and  solid 
media    as    well  as  on  plaster  blocks.     The 
temperature  limits  for  sporulation  on  plaster  blocks  are:    maximum, 
34-36°  C.,  minimum,  5°  to  10°  C. 

The  ascs  seem  to  form  indifferently  in  the  yeast  cells  or  at  the 
expense  of  some  cell  in  the  mycelium.  They  usually  enclose  a  single 
ascospore,  rarely  two.  The  ascospores  are  ellipsoidal  and  have  the 

1  Klocker,  A.  L'Endomyces  Javanensis,  n.  sp.  Comp.  Rend.  Trav.  du  Lab. 
de  Carlsberg.  8,  Book  4,  1909. 


nensis. 

Oidia,  Yeasts  and  Ascs.  — 

A.   Ascospores   in   Process 

of     Germination      (after 

Klocker). 


374  FUNGI   RELATED  TO  THE  YEASTS 

shape  of  a  slightly  flattened  ball.  They  have  but  a  single  membrane. 
This  membrane  is  supplied  with  more  or  less  distinct  elevations.  A 
projecting  ring  runs  around  the  middle  and  makes  the  ascospores  look 
like  those  of  Willia  Saturnus.  However,  this  ring  may  be  so  placed 
that  the  ascospores  look  like  hats.  Germination  of  the  ascospores  is 
accomplished  by  budding  in  the  yeasts  and  by  the  formation  of  ger- 
mination tubes  (Fig.  161,  A).  In  old  cultures  in  must,  there  is  formed 
on  the  surface  of  the  medium  and  along  the  walls  a  thick  ring  which 
may  grow  and  entirely  cover  the  surface.  The  least  jarring  of  the 
flask  causes  this  to  fall  to  the  bottom.  The  giant  colonies  on  gelatin 
have  a  viscous  appearance.  The  surface  is  much  folded  and  the  center 
slightly  sunken  by  a  slight  liquefaction  of  the  gelatin.  This  species 
does  not  invert  saccharose  or  ferment  dextrose;  it  acts  feebly  on 
levulose. 

Endomyces  javanensis  constitutes  a  more  immediate  link  between 
the  Saccharomycetes  and  the  Endomycetes  than  some  of  the  other  Endo- 
myces. The  fact  that  the  ascs  form  often  from  yeast  cells  unites  it 
very  much  more  to  the  Saccharomycetes  and  accordingly  it  is  advisable 
to  class  it  with  this  family. 


ENDOMYCES   CRUZI.     Mello  and  Paes  * 

Hello  and  Paes  found  a  yeast  with  oval  cells  (4-8  by  2-4  M)  in 
the  lungs  of  a  man  of  45  years  who  had  been  asthmatic  for  10 
years.  This  yeast  grew  well  on  glycerol  and  plain  potato,  Raulin's 
solution,  Sabouraud's  gelatin,  plain  and  glycerol  bouillon  and  alkalin 
agar.  On  solid  media  the  growth  was  a  yellowish  creamy  white. 
The  growth  was  more  abundant  on  alkalin  media.  This  yeast  fer- 
mented glucose,  maltose,  dextrose  and  saccharose.  In  cultures  these 
authors  observed  both  budding  forms  and  mycelial  cells.  The  ascs 
contained  from  two  to  four  spores.  This  fungus  seemed  to  be  closely 
related  to  Endomyces  vuillemini,  Landrieu,  1912.  The  latter  fungus, 
however,  prefers  an  acid  medium  and  does  not  ferment  dextrose. 

1  Mello,  F.  de  and  Paes,  A.  Endomyces  cruzi  n.  sp.  agent  (?)  d'une  endomy- 
cose  bronchique  simulant  1'asthme.  Arquivos  de  Higiene  e  Patologia  ex6ticas,  6 
(1918)  51-60.  Bull.  Past.  Inst.  17  (1919)  636. 


MONILIA   CANDIDA 


375 


Fig.  162.  —  Monilia  Candida 
Young  Cells  in  a  Scum. 


MONILIA  CANDIDA.    Bonorden l 
Syn.:    MONILIA  BONORDENH.     Vuillemin 2 

This  species  was  described  by  Hansen,3  and  was  isolated  from  fresh 
dung  and  fruit  juices  in  which  it  forms  a 
white  layer.  When  put  into  wort,  there 
is  abundant  growth  of  cells  having  the 
appearance  of  yeasts  and  resembling  S. 
ellipsoideus  and  cerevisiae.  (Fig.  162.) 
A  strong  alcoholic  fermentation  is  set  up 
during  which  the  surface  of  the  liquid  is 
covered  by  a  thick  scum;  this  is  made 
up  of  ordinary  cells  which  elongate  to  make  a  mycelium.  (Fig.  163.) 
According  to  the  investigations  of  Hansen,  this  fungus  forms  1.1  per 
cent  of  alcohol  by  volume  during  the  time  that  Saccharomyces  cerevisiae 
forms  6  per  cent.  But  while  Saccharomyces  cerevisiae  stops  at  this 
per  cent,  Monilia  Candida  continues  its  action.  After  6  months  fer- 
mentation there  is  5  per  cent  of  alcohol  by  volume. 
This  yeast  secretes  invertase  but  it  remains  in 
the  interior  of  the  protoplasm  and  never  diffuses 
through  the  membrane.  Fischer  and  Lindner  have 
found  that  it  is  impossible  to  extract  this  enzyme. 
They  have,  however,  inverted  saccharose  with  the 
dried  fungus,  even  in  the  presence  of  antiseptic  sub- 
stances. Cells  broken  up  with  glass  were  also  used. 
Monilia  Candida  inverts  maltose  and  ferments  the 
dextrine  (Bau).  It  easily  withstands  high  tempera- 
Fig.  163.  —  Monilia  turesr  On  account  of  this  it  may  develop  in  solu- 
mentous  Forms  in  tions  of  saccharose  and  beer  wort  at  40°  C. 


a  Scum  (after  Han- 
sen). 


Anderson  has  recently  mentioned  the  frequent 
presence  of  a  yeast  resembling  Monilia  Candida  in 
the  human  intestinal  tract.  Lindner  and  Knuth  have  also  found 
Monilia  Candida  in  epizootic  lymphangitis. 

1  The  genus  Monilia,   created  by  Persoon,  is  quite  badly  characterized.     It 
includes  filamentous  fungi  characterized  by  the  formation  of  oval  conidia,  ellip- 
tical or  in  chains  (conidial  yeasts  or  oidial  forms) . 

2  Vuillemin,  P.     Difference  fondam.  entre  le  genre  Monilia  et  les  genres  Scopu- 
lariopsis,  Acmosporium  et  Catenularia.     Bull.  Soc.  Mycol.  de  France,  27,  1911. 

3  Hansen,    E.    C.     Recherches   sur   la  physiologic  et   morphologie   des   alcoo- 
liques  ferments.     VII,  Action  des  ferments  alcooliques  sur  les  diverses  especes  de 
sucre.     Levures    alcooliques    a   cellules    resemblant    a   des    Saccharomycetes.     C. 
R.  Trav.  du  lab.  de  Carlsberg,  2,  Book  5,  1888. 


376 


FUNGI   RELATED   TO  THE   YEASTS 


MONILIA  NIGRA.    Browne 

This  Torula  was  isolated  by  Browne  from  raw  sugar.     One  sample 
of  such  sugar,  which  had  been  sealed  for  three  years,  gave  1500  colonies 


.          •• 


Fig.  163-A.  —  Small  Colonies  of  Monilia  nigra  in  Various 
Stages  of  Growth  (after  Browne). 


Fig.  163-B.  —  Magnified  Cells  of  Monilia  Nigra. 

Below  is  the  End  of  One  of  the  Hyphae,  Covered  with  Bud-Cells, 
and  Terminating  in  a  Cluster  of  Dark  Conidia. 

in  1  gram.  This  is  one  of  the  most  destructive  organisms  found  by 
this  author  in  Cuban  raw  sugar.  Browne  describes  the  colonies  on 
raw  sugar  agar  as  being,  at  first,  small  star-shaped  dots  which,  under 

1  See  reference  for  Torula  communis. 


MONILIA   NIGRA 


377 


the  microscope,  consists  of  radial  hyphae.  "  The  latter  throw  off  a 
conglomerate  of  bud  cells,  the  mass  of  which  increasing  in  thickness 
soon  gives  the  colony  a  starfish  appearance.  This  primary  growth 
is  usually  succeeded  by  a  secondary  growth,  due  to  the  propagation 


Fig.  163-C.  —  Magnified  Colony  of  Monilia  fusca. 

The  Radiating  Hyphae  are  covered  with  Bud-Cells  and  Dark  Conidia  (after  Browne). 

of  the  bud  cells,  which,  without  the  formation  of  hyphae,  germinate 
like  yeast  and  cover  the  center  of  the  colony  with  a  white  amoeba- 
like  film."  When  the  colonies  have  attained  a  diameter  of  from  1  to 
15  mm.,  the  hyphae  break  up  into  clusters  of  dark  conidia  which  give 


Fig.  163-D.  —  Magnified  Cells  of  Monilia  fusca. 

In  the  middle  is  a  branched  part  of  the  mycelium  bearing  4  bud-cells;  two  of  the 
latter  (one  germinating)  are  shown  at  the  left,  i  At  the  right  is  the  end  of  one  of 
the  hyphae,  breaking  up  at  the  end  into  3  conidia  and  in  the  middle  into  2 
oidia  (after  Browne). 

the  colony  a  black  color.  This  gives  it  the  name  of  Monilia  nigra. 
If  the  colony  stops  growing  before  the  conidial  stage  is  reached,  no 
black  color  is  assumed  but  the  white  remains.  Under  the  microscope, 
the  hyphae  are  of  the. ordinary  branched  type  but  more  often  are 
studded  with  clusters  of  bud-cells.  These  latter  are  elliptical  in  shape 
and  may  produce  new  hyphae,  or  propagate  like  a  yeast.  When  the 


378  FUNGI  RELATED  TO  THE  YEASTS 

hyphae  are  mature,  they  break  up  at  the  ends  into  thick- walled 
conidia.  The  disintegration  of  the  hyphae  into  thick-walled  cells 
may  also  occur  at  other  places  than  the  end.  This  gives  them  the 
appearance  of  oidia.  The  various  cell  units  of  this  microorganism 
contain  many  oil  globules.  This  Monilia  grows  well  in  raw  sugar 
solutions  except  those  which  are  most  concentrated.  The  culture 
fluid  becomes  turbid  with  mycelium  which,  after  several  days,  may 
extend  up  the  sides  of  the  tube.  There  is  slight  gas  formation  with 
a  fruity  odor.  The  action  on  the  raw  sugar  consists  principally  in 
the  inversion  of  sucrose.  This  inverting  ability  is  restrained  by 
raising  the  concentration  of  the  raw  sugar.  No  further  description 
of  this  organism  is  given  by  the  author. 

MONILIA  FUSCA.1    Browne 

The  colonies  of  this  Monilia  are  described  by  Browne  as  being  simi- 
lar to  those  of  Monilia  nigra  except  that  the  hyphae  are  much  longer, 
show  a  less  pronounced  tendency  to  form  the  yeast-like  structures,  and 
have  a  greenish  brown  color,  instead  of  black,  in  the  conidial  stage. 
The  Monilia  grows  in  raw  sugar  solutions  except  the  most  concen- 
trated. The  media  become  turbid  with  a  deposit  of  mycelium  and 
cells.  The  walls  of  the  container  to  a  distance  of  2  cm.  may  be  covered 
with  a  dark  conidial  growth.  There  is  a  slight  formation  of  gas 
and  a  fruity  odor.  Monilia  fusca  possesses  a  stronger  inverting  action 
than  Monilia  nigra.  Browne  regards  these  Monilia  as  the  most 
destructive  organisms  found  in  raw  sugar  on  account  of  their  ability 
to  adapt  themselves  to  different  conditions  in  their  environment. 

GEIGER'S  PSEUDOMONILIA  2 

Under  the  name  of  Pseudomonilia,  Geiger  has  included  a  number 
of  yeasts  which  will  be  described  at  this  time. 

Pseudomonilia  albomarginata 

The  cells  of  this  yeast  are  oval  (4  to  6  JJL)  and  have  a  protoplasm 
containing  one  or  three  refractive  granules  and  a  vacuole  containing 
crystals.  The  mycelium  is  made  up  of  long  filaments.  This  yeast 
forms  a  folded  scum.  The  giant  colonies  possess  special  forms  and  do 
not  liquefy  gelatin.  This  species  ferments  dextrose  slightly,  also  levu- 
lose  and  saccharose,  and  produces  a  slight  increase  in  the  acid  content 
of  solutions. 

1  See  reference  for  Torula  communis. 

2  Geiger,  A.    Beitrage  zur  Kenntniss  des  Sprosspilze  ohne  Sporenbildung.    Cent. 
Bakt.,  Abt.  2,  1910,  11. 


MONILIA  VINI  379 

Pseudomonilia  rubescens 

The  young  cells  are  oval  (3  to  5  /z  in  diameter)  in  the  beginning 
with  filamentous  mycelium.  The  scums  are  quite  thick  with  more 
or  less  marked  red  color.  The  giant  colonies  possess  a  faint  red 
color.  This  yeast  ferments  dextrose  in  an  active  manner.  It  is  very 
sensitive  to  the  action  of  lactic  or  tartaric  acid. 

Pseudomonilia  mesenterica 

The  cells  are  round  (3  to  6  /z  in  diameter)  and  often  elongated. 
Old  cells  contain  numerous  fat  droplets.  The  scum  is  thick  and 
strongly  folded.  The  giant  colonies  grow  rapidly  and  are  abundant. 
This  species  ferments  levulose. 

Pseudomonilia  cariilaginosa 

The  cells  are  oval,  from  5  to  6  /j  in  diameter,  pointed  at  both 
ends  and  enclosing  crystals  in  the  vacuoles.  They  are  intermingled  in 
a  filamentous  mycelium  with  cross  walls.  The  walls  of  the  cells  are 
mucilaginous.  The  scum  possesses  a  cartilaginous  appearance.  The 
giant  colonies  have  a  verrucose  aspect  and  the  gelatin  is  quite  rapidly 
liquefied.  This  species  ferments  saccharose,  but  scarcely  acts  on 

dextrose  and  levulose. 

« 

MONILIA  VINI.    Osterwalder1 

This  yeast  possesses  a  very  active  fermenting  function  and  acts 
like  a  bottom  yeast.  It  causes  a  more  active  fermentation  than  the 
other  Monilia  that  are  known.  It  does  not  stop  developing  in  the 
presence  of  very  large  amounts  of  acid  in  the  medium  in  which  it  is 
growing.  It  develops  well  in  solutions  with  4  per  cent  of  alcohol, 
especially  in  wine.  It  forms  secondary  products  such  as  volatile  and 
malic  acids.  Mycoderma  vini  possesses  a  less  active  fermenting  ability 
than  ordinary  wine  yeasts.  It  has,  however,  a  favorable  influence  on 
the  wine  but  produces  a  secondary  fermentation  of  sugar  which  has 
not  been  transformed  by  other  yeasts  in  the  primary  fermentation. 
It  does  not  contribute  a  bad  or  disagreeable  taste  to  the  wine. 

It  ferments  dextrose  and  levulose  especially,  and  saccharose, 
lactose  and  galactose  less  actively.  Maltose  is  very  feebly  attacked. 
It  seems  to  possess  a  soluble  sucrase  which  distinguishes  it  from 
Monilia  Candida.  The  giant  colonies  on  gelatin  exhibit  borders  with 
well-developed  fringes.  In  liquid  media  the  species  vegetates  at  first 

1  Osterwalder,  A.  Ein  neue  Garungsmonilia,  Monilia  vini,  n.  sp.  Cent.  Bakt. 
Abt.  II.  1912. 


380  FUNGI   RELATED  TO  THE  YEASTS 

in  the  form  of  a  sediment  and  later  produces  floes  in  the  liquid  and  a 
scum  like  that  of  a  mold.  The  sediment  consists  of  yeasts  about  six 
microns  in  length.  The  floes  and  scum  are  made  up  of  a  typical 
mycelium  with  numerous  yeasts. 


PARENDOMYCES  PULMONALIS.    Plaut 

This  yeast  was  isolated  by  Mantner  1  from  sputum  of  a  little  girl 
attacked  by  bronchitis.  In  the  sputum  it  exists  in  the  form  of  fila- 
ments and  conidial  yeasts.  In  cultures  the  fungus  does  not  give 
these  yeast  structures,  except  there  is  abundant  formation  of  mycelia. 
The  fungus  seems  very  closely  related  to  Monilia  Candida.  Plaut  has 
made  a  special  genus  related  to  Endomyces,  the  genus  Parendomyces. 

Senez2  described  a  fungus  very  much  like  Endomyces  albicans  which 
was  isolated  from  the  lungs  of  a  patient  believed  to  be  suffering  from 
tuberculosis.  White  granules  composed  of  fine  filaments  were  ob- 
served in  the  lesions.  When  these  granules  were  inoculated  into 
media  they  developed  into  creamy  white  patches.  Carbohydrate 
media  and  carrot  slants  seemed  to  be  best  adapted  for  growth.  In 
liquid  cultures  there  was  abundant  growth  provided  the  media  were 
not  acid  in  reaction.  The  organism  was  a  strict  aerobe.  Both 
round  and  filamentous  forms  were  reported.  The  round  cells  mul- 
tiplied by  budding.  The  buds  were  able  to  break  off  and  reunited 
end  to  end  to  form  filaments.  Ascs  appeared  in  old  cultures  on 
gelatin.  They  were  large  and  oval  in  shape  and  about  10  fj,  in" 
diameter.  Senez  distinguished  this  fungus  from  Endomyces  albicans 
by  the  appearance  of  the  lesions  in  the  lungs,  its  dislike  for  acid 
media,  the  ease  of  cultivation  on  alkaline  media,  the  difference  in 
appearance  on  solid  media  and  its  almost  negative  pathogenicity 
when  demonstrated  experimentally. 

1  Mantner,  H.    Parendomyces  pulmonalis,  Plaut  eine  bisher  nicht  beschriebene 
Monilia-art.     Cent.  Bakt.  Abt.  I,  74  (1916),  203. 

2  Senez,  A.     El  Endomyces  pulmonalis.     Boletin  del  Labor,   de  Bacter.  de 
Tucuman  (Rep.  Argentina)  1  (1918),  58-80.    Bull.  Past.  Inst.  17  (1919),  636. 


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BIBLIOGRAPHICAL  INDEX  401 

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402  BIBLIOGRAPHICAL  INDEX 

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INDEX  OF  NAMES 


ACHALME,  121,  270,  363 

Adametz,  60,  308 

Aderhold,  243,  334 

Albert,  90 

Alwood,  164 

Amato,  55 

Anderson,  124,  220,  318,  319,  320,  :  21, 

341,  364,  365,  375 
Ando,  323 
Antoni,  91 
Artari,  59,  254 
Arthault,  353,  355 
Aruch,  346,  348 
Asai,  342 
Ashford,  364,  365 
Auld,  65 

BABES,  40 

Bachmann,  130,  148 

Bail,  231 

Bainier,  318 

Balbaini,  127 

Barker,  19,  20,  21,  115,  194,  206,  219, 

250,  302. 

Bary,  2,  4,  13,  137 
Batschniskaia,  260 
Bau,  65,  66 
Baudrexel,  124 
Bay,  245 
Beauverie,     40,     52,     167,     355,     356, 

363 

Bectoamp,  4 
Behrens,  229 
Behring,  42 
Beijerinck,  18,  46,  59,  67,  69,  126,  175, 

180,  181,   197,   198,  202,  272,  283, 

285,  300,  307,  314 
Beinarcki,  184 
Bellamy,  85 
Belohoubek,  56 
Benorden,  375 
BSrard,  84 
Berlese,  135 
Bernard,  89 


Berthelot,  89,  102 

Beurmann,  de,  345,  346,  359,  351,  358, 

384 

Bilewsky,  316,  317 
Binot,  121,  271 
Bitto,  45 
Bizzozero,  353 
Blackmann,  104 
Blanchard,  121,  271,  353 
Boas,  110 

Bocchichio,  59,  60,  175,  310 
Bokorny,  54,  55,  60,  66,  70,  71,  75,  79, 

110 

Bonorden,  375 
Boquet,  346,  347 
Bottcher,  156,  159 
Bouffard,  88 
Bourn,  37,  160,  161 
Boullinger,  59,  69,  77 
Boussingault,  69 
Boutroux,  136,  256 
Boyen-Jensen,  96,  104 
Bra,  131,  355 
Brazolla,  351 
Brebeck,  107,  285,  340 
Brefeld,  4,  13,  83,  137,  158 
Bresson,  65 
Brewer,  359 
Bride,  356 
Bronfenbrenner,  162 
Brown,  88 

Browne,  309,  329,  376,  377,  378 
Bruschi,  78 
Brusendorf,  336 
Buchner,  4,  56,  58,  60,  88,  89,  90,  91, 

92,  93,  94,  96 
Buchta,  109 
Burri,  311 
Buscaloni,  230 
Busse,  270,  348,  352 
Biitschli,  118 

CAGNARD-LATOUR,  3 
Carpano,  126 


411 


412 


INDEX   OF   NAMES 


Casagrandi,  4,  12,  45,  123,  230,  355 

Castellani,  360,  363,  364 

Cattaneo,  353 

Cavara,  230 

Cesari,  23 

Chamber-land,  132,  136 

Chatton,  20,  292 

Chopin,  111 

Clautriau,  54 

Cienkowski,  332 

Classen,  71 

Clausen,  299 

Clerc,  354 

Cochrun,  108 

Cohn,  F.,  149,  154,  313,  317 

Cohn,  E.,  307,  352 

Collau,  338 

Conte,  127 

Corselli,  121,  352 

Constantin,  351,  352 

Coupin,  306 

Cremer,  62,  77 

Csizer,  78 

Culex,  118 

Curtis,  121,  270 

Czapski,  65 

Czier,  80 

DAIEREUVA,  363 

Dangeard,  37,  40,  49,  118,  360 

Dantec,  121,  357 

Delbruck,  91,  100,  101,  124,  233 

Demanche,  356 

Demme,  123,  355 

Demolon,  75,  89,  103 

Denamur,  241 

Denys-Chochin,  83,  89 

Desm,  331 

Devaux,  102 

Diehl,  60 

Dienert,  185 

Bold,  353 

Dombrowski,   142,  210,  226,  266,  311, 

312,  313,  338,  369,  370,  372 
Dubourg,  185 
Dubrunfaut,  85 
Duchacek,  92 
Duclaux,  60,  64,  69,  84,  89,   103,  107, 

175,  307,  336 
Diiggeli,  311 
Dumas,  53 
Duval,  358 


ECHON-ECHEUG,  346 

Effront,  61,  72,  105,  184 
Ehrenberg,  158 
Ehrlich,  71,  72,  74,  89 
Eischenschitz,  37 
Elion,  68 
Emberg,  112 
Emmerling,  87 
Engel,  4,  151,  272,  332 
Errera,  54,  76 
Escherich,  352 
Eijkmann,  202 
Euler,  112,  164 

FABRONI,  3 

Farber,  66 

Faucheron,  127 

Felice,  san,  121 

Fermi,  346,  348 

Fernbach,  67,  68,  69,  96,  128 

Fischer,  B.,  285,  340 

Fischer,  E.,  61,  65,  87,  107,  375 

Fisco,  121 

Flava,  360 

Flemming,  161 

Freire,  360 

Freudenreich,  125,  147,  175,  307 

Fresenius,  316,  317 

Frisco,  121,  351 

Fromherz,  70 

Fuhrmann,  48 

Funk,  129 

GAY-LUSSAC,  88,  100 

Cayon,  108 

Geerlings,  247 

Geiger,  378 

Geret,  59,  62 

Giddings,  111 

Gilchrist,  345,  346] 

Gisevi,  343 

Giurel,  109 

Goetano,  360 

Goldewsky,  85,  102 

Gorodkowa,  152,  214 

Gotti,  351 

Gougerot,    345,    346,    348,    351,    358, 

359 

Gradenigo,  360 
Greg,  201,  233 
Gregoriew,  59,  91,  92 
Grehant,  80,  81 


INDEX   OF   NAMES 


413 


Griaznoff,  98 

Gromow,  59,  91,  92 

Gronlund,  C.,  246,  299 

Grossbusch,  323 

Grotenfelt,  175,  265 

Grouven,  111 

Griiss,  66,  77,  78,  81,  94 

Guegen,  120,  122,  126,  176,  350,  353 

Guiart,  J.,  122,  271 

Guilliermond,  9,  16,  20,  21,  22,  24,  27, 
34,  36,  37,  42,  49,  51,  77,  133,  138, 
139,  152,  160,  198,  200,  201,  210, 
211,  216,  217,  221,  224,  228,  230, 
257,  260,  261,  262,  280,  282,  328, 
339,  355,  362,  363,  367,  368,  370 

HAHN,  59,  62 

Hallier,  73 

Hansen,  2,  4,  6,  8,  9,  10,  13,  23,  29,  30, 
33,  35,  83,  106,  109,  112,  113,  114, 
115,  117,  118,  134,  135,  136,  147, 
148,  151,  153,  155,  159,  164,  168, 
169,  171,  172,  178,  182,  184,  185, 
186,  187,  189,  227,  228,  231,  234, 
237,  238,  240,  252,  254,  273,  275, 
276,  283,  284,  293,  316,  323 

Harden,  91,  96,  97,  185 

Harrison,  311 

Harter,  359 

Hartmann,  301 

Hawk,  124,  129 

Haydruck,  68,  72,  74,  91,  123,  128, 
149 

Heidenhain,  160. 

Heinze,  307 

Heinzelmann,  70 

Henneberg,  42,  66,  78,  333 

Henneguy,  127 

Henry,  65 

Herwerden,  van,  40,  41 

Herzog,  118 

Hest,  van,  Heuse,  78 

Heymons,  127 

Hieromymous,  Hinsberg,  54 

Kite,  111 

Hoehn,  92 

Hoffmann,  71,  92 

Hoffmeister,  16 

Holm,  180,  200,  248,  264,  302 

Hollande,  305 

Hosaceus,  62,  77 

H6ye,270,306 


Hudelo,  358 
Hunter,  64,  175 
Huxley,  127 

INUI,  302 
Iraklionoff,  83 
Irmisch,  234 
Isrealsky,  74 
Issajew,  66 

JACQUEMIN,  109,  243 

Janssens,  37,  49,  315,  316 

Jensen,  61,  175,  210,  266,  313,  338 

Jodin,  73 

Johnson,  304 

Jones,  56,  61,  72 

Jorgensen,  132,  186,  200,  233,  243,  248, 

258,  264,  276,  304,  314,  315 
Jiihler,  132 

KALANTHAR,  64,  313 

Karczag,  64 

Kayser,  69,  70,  75,  77,  89,  103,  108,  243, 
250,  264,  309,  337 

Khoury,  126,  310,  337 

Kinokotin,  22,  191,  222,  223 

Kita,  78,  330 

Klatte,  92 

Klebahn,  133 

Klebs,  114 

Klocker,  20,  26,  33,  34,  107,  115,  118, 
133,  143,  163,  168,  170,  185,  189, 
193,  194,  206,  221,  222,  225,  252, 
274,  275,  280,  281,  288,  323,  325, 
326,  367,  369,  373 

Kluyver,  76,  78 

Knuth,  375 

Koch,  77,  155 

Kohl,  41,  62,  77,  78 

Kolodiziejaka,  74 

Kosai,  247,  284 

Koser,  72 

Kossel,  41,  56 

Kossowicz,  67,  70,  71,  73,  110 

Kostytschew,  104 

Kozai,  247,  284 

Kramer,  321 

Krueger,  265 

Kruis,  89 

Kruyff,  de,  20,  106,  207,  302,  323 

Kullberg,  78 

Kusserow,  72,  99,  104 


414 


INDEX   OF  NAMES 


Kut,  98 

Kiitzing,  3,  158,  196,  231 

LABOK  DE,  69,  103 

Laer,  van,  124,  234,  241 

Landrieu,  374 

Lanzenberg,  69 

Lasche,  256,  331,  340 

Lasseur,  273 

Laurent,  69,  75,  77,  149,  361 

Lebedeff,  90,  96,  97 

Leberle,  176,  331 

Leblanc,  37,  49 

Lechartier,  85 

Lecomier,  156 

Legrain,  121,  271 

Lenhossek,  161 

Lepeschkin,  179,  199 

Lesieur,  304,  355,  356,  363 

Leeuwenhoek,  3 

Levene,  57 

Liebermann,  45 

Lindau,  196,  343,  344 

Linder,  71,  72 

Lindner,  8,  27,  72,  73,  75,  76,  77,  78,  79, 
87,  111,  126,  143,  153,  157,  159,  160, 
161,  164,  166,  174,  176,  180,  189, 
194,  199,  211,  215,  226,  225,  233, 
248,  273,  274,  277,  279,  287,  299, 
314,  324,  344,  370,  371,  375 

Linne,  3 

Linossier,  89,  362 

Lipman,  73 

Lister,  154 

Lob,  99 

Loederich,  358 

Lohnis,  73 

Low,  66 

Lubhock,  127 

Lucet,  120,  349 

Ludwig,  106,  227 

Lugol,  161 

Lutz,  125,  176,  279 

MAFFUCI,  122,  350 
Maggiora,  360 
Malasses,  353 
Mangin,  45,  304 
Mantner,  380 
Marchand,  245 
Marcone,  346,  348 
Marpmann,  328 


Marx,  243 

Mangenot,  366 

Maummann,  73 

Mayer,  68,  69,  70,  72,  148 

Maze,  85,  102,  175,  313 

McKim,  129 

Melard,  283 

Mello,  374 

Melsens,  111 

Meigen,  45 

Meisenheimer,  55,  74,  94,  95 

Meissl,  164 

Meissner,  77,  107,  299 

Mercier,  360 

Mertens,  315,  316 

Metschnikoff,  34,  118,  126,  289,  290 

Meyen,  195,  231 

Meyer,  68,  111 

Micellone,  118,  346,  348 

Mitra,  110 

Mitsuda,  209 

Mitscherlick,  158 

Muller-Thurgau,  135,  243 

Muntz,  69,  85 

NADSON,  22,  146,  183,  191,  194,  222 

Naegeli,  118,  149 

Nakayaza,  203,  204 

Nakazawa,  250,  251 

Nastjukow,  243 

Negre,  346,  347 

Nessler,  67 

Neubauer,  70 

Neuberg,  64,  98,  99 

Neumann,  66 

Neuss,  54 

Neville,  55 

Nielsen,  118,  228,  276 

Nikolajewa,  311 

Nishimura,  209,  216 

Norris,  185 

Nowak,  111 

OLSEN-SOPP,  268 
Orsos,  173 

Osterwalder,  243,  379 
Oudemans,  353 
Owen,  254,  309 

PACOTTET,  133,  134 
Paes,  374 
Palladine,  83 


INDEX   OF   NAMES 


415 


Pasteur,  3,  4,  69,  82,  84,  85,  88,  89,  100, 
101,  132,  147,  148,  154,  182,  235, 
332 

Payen,  90 

Pearce,  20,  21,  219,  249,  302 

Peglion,  196,  290 

Pekelharing,  353 

Peniston,  37 

Penau,  362 

Perenyi,  160 

Perkins,  108 

Perrier,  103 

Persoon,  90,  196,  330 

Pichi,  277 

Piedallu,  61 

Pierantoni,  127,  128 

Pierantoni,  313 

Pillai,  73 

Plaut,  361,  380 

Plimmer,  121,  351 

Polezenius,  85,  102 

Potron,  263 

Preyer,  269 

Pringsheim,  H.,  61 

Pringsheim,  F.,  72,  316,  317 

Prinsen-Geerligs,  247 

Purvis,  115,  119 

QUINQUAD,  80,  81,  361 

RABINOWITCH,  122 

Rajat,  363 

Rapp,  90 

Raulin,  149,  336,  356,  359,  362,  374 

Raynaurd,  120,  349 

Read,  56 

Recklinghausen,  von,  109 

Reess,  2,  4,  13,  111,  152,  231,  241,  244, 

254,  269,  273,  323,  332,  361 
Regnard,  111 
Reinicke,  234 
Reinfirths,  98 
Renarck,  118,  230 
Resenbeck,  92 
Rettger,  70,  354 
Rey-Pailhade,  66 
Riboni,  313 
Richter,  212 
Risler,  81 
Rist,  126,  310,  337 
Rivolta,  120,  346,  347,  348,  353 
Robin,  4,  230,  361 


Roeser,  89 

Rogers,  61,  313 

Roncali,  345 

Rose,  54,  106,  225,  252,  305,  363 

Rossi,  332,  342 

Rothenbach,  339 

Roux,  69,  89,  362 

Rubinski,  267 

Rubner,  80 

Rulke,  72 

Russel,  121 

SABOURAND,  347,  374 

Saccardo,  206,  245,  246,  353 

Saito,  K,  75,  78,  116,  118,  120,  143,  207, 

210,  212,  248,  251,  253,  255,  256, 

258,  279,  282,  284,  287,  301,  306, 

330,  335,  340,  366 
Saito,  R.,  20,  115 
Salkowski,  45,  105 
San  Felice,  346,  349,  350,  352,  360 
Sartory,  116,  273,  289,  317,  318,  354, 

356,  357 
Sauton,  75,  89 
Schipin,  267 
Schionning,  16,  143,  284,  299,  367,  368, 

370 

Schlesinger,  162 
Schlossberger,  45,  53 
Schneider,  291 
Schonfeld,  124 
Schroter,  189,  316 
Schulz,  72,  158 
Schutaemberger,  54,  56,  81 
Schwann,  3,  13 
Schwartz,  73,  121,  271 
Schwellengrebel,  48 
Seidell,  129 
Seiter,  133 
Senez,  380 
Sergent,  123 

Seynes,  de,  4,  13,  331,  332 
Shiga,  72 

Siefert,  243,  276,  278,  332 
Sirleo,  122,  350 
Skchiwan,  122 
Slator,  95,  158,  164 
'Sorel,  132 
Spitta,  91 
Spreng,  45 
Steinhaus,  357 
Stern,  68 


416 


INDEX  OF  NAMES 


Steuber,  285,  286 

Stockhausen,  78,  80 

Stokes,  345 

Stoklasa,  102 

Stoppel,  139 

Stressburger,  45 

Straughn,  61,  72 

Sulc,     12,    127,     128,    202,    205,    325, 

360 
Sydow,  206,  222 

TAKAHASHI,  20,  208,  215,  218,  219,  258, 

339 

Tammann,  111 
Tanner,  67 
Tan  ret,  45 
Thaon,  126,  349 
Thierfelder,  87,  61 
Thomas,  74,  72 

Tieghem,  van,  13,  61,  156,  189,  313 
Tokishige,  346,  347 
Tolomei,  66 
Trabut,  353 
Trecul,  4 
Trillat,  75,  89 
Troisier,  121,  270,  363 
Trommsdorff,  55 
Turpin,  231,  293 

UNNA,  161 

VANDEVELDE,  128 

Viala,  133 

Vines,  60 

Visselingh,  45 

Voelz,  124 

Voltz,  71 

Vries,  de,  180 

Vuillemin,  P.,  121,  271,  345,  346,  348, 


349,  350,  352,  353,  355,     61,  362 
363,  365,  375 
Vulquin,  67 

WAGNER,  37,  38,  77 

Ward,  109,  247 

Warwich,  115,  119 

Wasserzug,  152 

Waterman,  70 

Weakley,  111 

Wehmer,  83,  303,  305,  336 

Weigmann,  313 

Wells,  108 

Welter,  54 

Wender,  66 

Went,  247 

Weininger,  111 

Wildier,  130,  148 

Wilhelmi,  24,  230 

Will,  6,  8,  12,  45,  77,  78,  107,  111,  124, 
165,  176,  186,  233,  245,  246,  268, 
295,  296,  327,  329,  :  34,  335 

Williams,  130,  148 

Winogradsky,  331 

Woff,  73 

Wood,  359 

Wortmann,  100,  102,  136,  243,  331 

Wroblewsky,  62,  91,  92 

Wust,  71,  79 

YABE,  247,  322 

Young,  91,  96,  97 

Yukawa,  208,  215,  218,  219,  258 

ZALESKY,  74 

Zetlin,  116 

Zikes,  73,  110,  273,  274,  287,  323 

Zimmermann,  73 

Zopf,  269 


INDEX  OF  SUBJECTS 


ABSCESS,  yeast  from,  354 
Accessory  substances  in  yeasts,  129 
Adhesive  cultures,  Lindner,  159 
Affinities  of  yeasts,  139 
Agglutination  of  yeasts,  46 
Air,  influence  on  sporulation,  117 
Albuminoids  in  yeasts,  55 
Albumoses  in  yeast  nutrition,  69 
Alcohol,  determination,  method  for,  163 

formation,  by  Asp.  glaucum,  C4 

by  S.  Ludwigii,  229 

in  yeast  nutrition,  75 

source  of  carbon,  78 
Alcoholic  fermentation  by  molds,  84 
Alwood  fermentation  valve,  164 
Amino  acids  in  yeast  nutrition,  72 
Amins  in  yeast  nutrition,  70 
Ammonium,  salts  in  yeast  nutrition,  71 

sulfate  as  a  source  of  nitrogen,  80 
Amygdalase,  66 
Amylase,  62 
Angina,  yeast  from,  354 
Antiprotease,  in  yeast,  60 

in  yeast  juice,  93 
Antiseptics,  effects  on  yeasts,  111 
Arrack,  yeast  from,  202 
Ascospore,  detection  of,  159 

formation  temperature  limits,  168 

germination  in  asc,  35 

germination  of,  29 

staining,  method  for,  52 
Aspergillus  glaucum,  alcoholic  fermenta- 
tion, 84 
Atelosaccharomyces  Harteri,  359 

of  Brewer  and  Wood,  359 

of  Hudelo,  358 
Autolysis  of  yeasts,  104 
Autophagy  of  yeasts,  104 

BEAUVERIE'S  method  for  staining  asco- 

pores,  52 

Beer  wort,  preparation,  150 
Bees,  yeasts  on,  206 
Beijerinck's  test  for  iron,  314 
Torula,  300 


Biochemical  activity  of  yeasts,  174 

Bios,  130 

Black  tongue,  349 

Blastomyces  Hessleri,  354 

Boils,  yeast  treatment,  129 

Bottcher's  moist  chamber,  156 

Bottom  yeasts,  transformation  into  top 

yeasts,  186 

Bread  yeast,  efficiency  of,  163 
Brusendorf's  Mycoderma,  336 
Buchner's  zymase,  89 
Budding,  in  yeasts,  9,  47 

physiological  condition  of,  112 

temperature  relations,  167 

CANCER,  yeast  from,  345,  351,  352 
Carbohydrate,  enzymes,  61 

fermentation,  methods  for,  162 
Carbon,  alcohol  as  a  source  of,  84 

ammonium  sulfate  as  a  source  of,  80 
Carboxylase,  65 
Carcinoma,  349 

Carrot  media  for  sporulation,  152 
Casease  in  yeasts,  59 
Catalase,  66 
Cell,  condition  and  sporulation,  115 

division,  9 

Cellulose  in  membrane,  45 
Chamber-land  flask,  147 
Changes  in  cell  during  fermentation,  46 
Changing,  molds  into  yeasts,  133 

yeasts  into  molds,  133 
Characteristics  of  sediment,  167 
Cheese,  Lombard,  yeast,  310 

yeast  from,  307 
Chemical,  composition  of  yeasts,  53 

equation  of  alcoholic  fermentation,  88 
Chinese  yeast,  212,  282,  366 
Cider,  yeasts  from,  302 

yeast  of  Pearse  and  Barker,  249 
Classification  of  yeasts,  189 
Coagulation  of  milk,  60 
Coccidiascus  Legeri,  292 
Coenzyme,  91 
Coferment,  91 


417 


418 


INDEX  OF  SUBJECTS 


Cohn's  solution,  149 
Composition  of  yeasts,  53 
Conditions  necessary  for  alcoholic  fer- 
mentation, 81 
Copulation,  of  ascospores,  23 

preceding  asc  formation,  15 
Counting  cells  in  cultures,  157 
Cryptococcus,  agregatus,  321 

anobii,  352 

Bainieri,  318 

Capillitii,  353 

Cavicola,  353 

Corsellii,  351 

de  Gotti  and  Brazzola,  351 

degenerans,  345 

farciminosus,  348 

general  characteristics,  196 

Gilchristi,  345 

glabratus,  320 

granulomatogenes,  350 

Guilliermondi,  355 

hominis,  348 

hominis  costantini,  352 

Kleinii,  352 

Lesieuri,  356 

linguae-pilosae,  349 

lithogenes,  349 

neoformans,  354 

niger,  350 

of  Clerc  and  Sartory,  354 

ovalis,  353 

ovoideus,  320 

parasitaris,  353 

Plimmeri,  351 

psoriaris,  353 

Rogeri,  356 

Ruber,  355 

salmoneus,  357 

sulfureus,  356 

Tokishigei,  346 

verrucosus,  319 
Culture  of  yeasts,  147 
Cytological,  changes  during  multiplica- 
tion, 47 

sporulation,  48 
Cytology  of  yeasts,  37 
Cytoplasm  and  its  constituents,  38 

DAPHNIA,  yeast  from,  290 
Dauerhefe,  90 

Debaromyces,     general     characteristics, 
194 


Debaromyces  globosus,  characterization, 

221 

germination  of  ascospores,  33 
parthenogenetic  ascs  in,  26 
Debaromyces  tyrocola,  characterization, 

222 

copulation,  22 
Definition  of  yeasts,  1 
De  Kruyff's  Torula,  302 
Dematium  pullulans,  2 

fixation  of  nitrogen  by,  73 
Demonstration  of  glycogen,  161 
Dermatitis,  yeast  from,  345 

yeasts,  353 

Detection  of  ascospores,  159 
Determination  of  power  of  multiplica- 
tion, 157 
Development  of,  yeast  forms,  28 

yeasts,  Slator's  method,  158 
Dextrins  in  yeast  nutrition,  76 
Differences  in  fermenting  function,  85 
Dilution  method  for  separating  yeasts, 

154 

Dimensions  of  yeast  cells,  6 
Dinucleotides  in  yeasts,  56 
Dioxyacetone  in  alcoholic  fermentation, 

98 
Direct  germination  of  ascospores  in  ascs, 

35 

Disinfectants,  action  on  yeasts,  111 
Distribution  of  yeasts  by  insects,  136 
Division  of  nucleus  in  yeasts,  49 
Division  of  yeast  cells,  9 
Dombrowski's,  Mycoderma  from  milk, 

338 

Torula,  311 

Drop  culture  method,  157 
Duclaux's,  Torula,  307 

yeast,  336 
Durable,  ceUs,  12 

yeast,  90 
Duration  of  life  of  yeasts,  107 

EFFECT  of,  adrenalin  on  yeasts,  73 
antiseptics  on  yeasts,  111 
dextrose  on  sporulation,  115 
disinfectants  on  yeasts,  111 
humidity  on  sporulation,  119 
light  on  sporulation,  119 
light  on  yeasts,  109 
metals  on  yeasts,  110 
moisture  on  yeasts,  109 


INDEX  OF  SUBJECTS 


419 


Effect  of,  ozone  on  yeasts,  111 

pressure  on  yeasts,  111 

radioactive  emanations  on  yeasts,  109 

ultra  violet  light  on  yeasts,  109 
Efficiency  of  bread  yeast,  163 
Eidam  cheese,  yeast  from,  307 
Emulsin,  65 
Endomyces,  characteristics  of  genus,  361 

Albicans,  characteristics,  361 

capsularis,  367 

effect  of  pressure  on,  111 
Endomyces  Cruzi,  374 
Endomyces  javanensis,  373 

fibuliger,  370 

Hordei,  366 

Lindneri,  366 
Endospores,  13 
Endotryptase,  59 
Enzymes  for,  carbohydrates,  61 

polysaccharides,  62 

of  nucleo  proteins,  60 

of  yeasts,  58 

proteolytic,  59 

Esters  as  a  source  of  carbon,  79 
Ethylacetate  as  a  source  of  carbon,  78 
Existence  of  glycogen  in  yeast,  77 
Exoascus,  yeast  forms  of,  139 
Extent  of  alcoholic  fermentation,  84 

FAT  droplets  in  yeasts,  39 

Fats  in  yeasts,  54 

Fatty  acids  in  yeast  nutrition,  75 

Fermentability  of,  polysaccharides,  62 

trisaccharides,  62 
Fermentable  sugars,  86 
Fermentation,  as  a  phase  of  respiration, 
101 

in  moist  chambers,  162 

of  carbohydrates,  methods  for,  162 

valve,  Al woods,  164 

valve,  Meissl's,  164 
Figs,  yeasts  from,  107 
Fixation  of  nitrogen  by  yeasts,  73 
Flemming's  solution,  161 
Flocculation  of  yeasts,  46 
Fly,  yeast  on,  281 
Formation  of  ascospores,  13 
Formula  of  alcoholic  fermentation,  88 
Freudenreich's  kephir  yeast,  307 
Frohberg  yeast,  233 
Fruits,  yeast  from,  107 
Furunculosis,  use  of  yeasts  in,  129 


GAY-LUSSAC'S  theory  of  alcoholic  fer- 
mentation, 88 
General  characteristics,  5 

of  alcoholic  fermentation,  81 
Germination  of  ascospores,  29 
Giant  colonies,  174 
Ginger  beer,  yeasts  from,  206 
Glanders,  African,  yeast  in,  346 
Glucoside  enzymes,  65 
Glycogen,  76 

demonstration  of,  161 

purpose  of,  in  yeasts,  76 
Glycogenase,  62 
Gorodkowa's  medium,  152 
Grapes,  yeast  on,  243 
Growth  on  solid  media,  173 
Griiss'  theory  of  alcoholic  fermentation, 

95 
Guanase,  60 

HABITAT  of  yeasts,  106 
Hansenia  apiculata,  273 

general  characteristics,  195 

valbyensis,  275 
Hansen's,  flask,  165 

flask  for  culturing  yeasts,  151 

media,  148 

method  for  detecting  spores,  159 

Torula,  293 

Haydruck's  medium,  149 
Hemicellulose  in  yeast  membrane,  45 
Hemocytometer,  157 
Heterogamic  copulation,  21 
History  of  yeasts,  3 
Honey  yeast,  212 
Hoye's  Torula,  306 
Humidity,  effect  on  sporulation,  119 
Hydrocarbons  in  yeast  nutrition,  75 
Hydrogen  sulfid  formation,  67 
Hydrogenase,  66 

INFLUENCE  of  air  on  sporulation,  117 
Insects,  and  distribution  of  yeasts,  136 

yeast  from,  292 
Intestinal  yeast,  230 
Intramolecular  respiration,  84 
Inulase,  62 

Iron,  test  for  by  yeasts,  314 
Isolation  of  yeasts,  153 

JOHANNISBERG  yeast,  I,  II,  243 
II,  copulation  of  ascospores,  24 
II,  isogamic  copulation,  23 


420 


INDEX  OF  SUBJECTS 


KARYOKINESIS,  50 

in  Schizosaccharomyces  octosporus,  198 
Kayser's  yeast,  308 
Kefir,  yeasts  in,  125 
Kephir,  yeast  from,  307 
Klocker's  method  for  alcohol  determina- 
tion, 163 

Koumys,  yeast  from,  267 
Kramer's  red  Torula,  321 

LACTASE,  63 
in  yeast  juice,  94 

Lactic  acid  theory  of  alcoholic  fermenta- 
tion, 95 

Lactomyces  inflans  caseigana,  310 

Larvae,  yeast  from,  202,  205 

Laurent's  medium,  149 

Lebedeff's  theory  of  alcoholic  fermenta- 
tion, 97 

Leben,  126,  310 
yeast  from,  337 

Le  Dantec's  yeast,  357 

Lenhossek's  fixing  fluid,  161 

Life  cycles  of  yeasts,  145 
in  nature,  134 

Light,  109 

effect  on  sporulation,  119 

Lindner  and  Meissner's  Torula,  299 

Lindner's,  adhesive  culture,  159 

method  for  securing  pure  cultures,  157 

Lipase,  61 

Lipoids  in  yeasts,  55 

Lithium  salts,  effects  on  yeasts,  110 

Logos  yeast,  241 

Longevity  of  yeasts,  107 

Lungs,  yeast  from,  350 

Lymphangitis,  yeasts  in,  346 

MACROSCOPIC  appearance  on  solid  media, 

173 

Magnesium  in  yeast  nutrition,  68 
Malt  water,  preparation,  150 
Maltase,  63 

Maltose,  utilization  of,  76 
Mayer's  culture  medium,  148 
Mazun,  yeast  from,  311 
Medusomyces,    general    characteristics, 

196 

Medusomyces  Gesevii,  343 
Meissl  fermentation  valve,  163 
Melibiase,  62 
Melizitase,  62 


Membrane  of  yeasts,  45 
Mercier's  yeast,  360 
Metachromatic  granules,  39 

staining  of,  161 
Metachromatin,  39,  40 

and  sporulation,  42 
Metals,  effect  on  yeasts,  110 
Methods  of  securing  sporulation,  151 
a-methylglucase,  65 
Milk,  yeast  in,  307,  355 
Mineral  elements  in  yeast  nutrition,  68 
Mode  of  action  of  zymase,  94 
Moist  chamber,  156,  158 
Moisture  in  yeast  growth,  109 
Molasses,  yeast  from,  200 
Molds,  alcoholic  fermentation  by,  84 
Monilia  Candida,  375 
Monilia  fusca,  378 
Monilia  nigra,  376 
Monilia  vini,  379 

Monospora,  general  characteristics,  195 
Monospora  cuspidata,  290 

germination  of  ascospores,  34 
Morphological  variations,  178 
Mucilaginous  substances  in  yeasts,  45 
Mucor  mucedo,  alcoholic  fermentation 

by,  84 

Multiplication,  changes  during,  47 
Multiplication  power  of  yeasts,  157 
Mycoderma,  general  characteristics,  196 
Mycelial  formation  in  yeasts,  8 
Mycetocytes,  128 
Mycetomes,  128 
Mycoderma,  acidipani,  342 

cerevisiae,  331 

chavalieri,  339 

cucumerina,  334 

decolorans,  335 

duplex,  333 

of  Fischer  and  Brebeck,  340 
.  gallica,  334 

Henneberg,  333 

lebenis,  337 

monosa,  341 

pineapple,  from,  337 

rugosa,  341 

sp.,  340 
Mycoderma,  tannica,  342 

tenax,  333 

valida,  334 

vini,  331,  332 
Myrosinase,  66 


INDEX  OF  SUBJECTS 


421 


NADSONIA,  general  characteristics,  194 

elongata,  223 

fulvescens,  222 
Nematospora,  coryli,  290 

coryli,  germination  of  ascospores  in, 
34 

general  characteristics,  196 

lycopersici,  291 

Neuberg's  theory  of  alcoholic   fermenta- 
tion, 98 

Nitrogen  fixation  by  yeasts,  73 
Nitrogenous  compounds,  in  yeasts,  55 

in  yeast  nutrition,  69 
Noegeli's  medium,  149 
Nuclear  fusion  in  yeasts,  49 
Nucleases,  60 
Nucleic  acid  in  yeast,  57 
Nucleus,  staining  of,  160 

of  yeasts,  38 
Nutrition  of  yeasts,  68 
Nuts,  yeast  from,  290 

ORIGIN  of  yeasts,  132 

Oxidizing  enzymes,  66 

Oxygen,  influence  on  sporulation,  117 

Oxy malic  acid,  fermentation  of,  63 

Oxynitrilase,  66 

Ozone,  effect  on  yeast,  111 

PARASACCHAROMYCES,  ASHFORDII,  364 

Thomasii,  365 
Parasitism  in  yeasts,  120 
Parendomyces  pulmohalis,  380 
Parthenogamy,  23 
Parthenogenesis,  25 
Pasteur  flask,  147 
Pasteur  medium,  148 
Pathenogenicity  of  yeasts,  120 
Pasteur's  theory  of  fermentation,  99 
Pearse  and  Barker's  Torula,  302 
Peptones  in  yeast  nutrition,  69 
Perenyi's  solution,  161 
Periostitis,  yeast  from,  348 
Permanent  variations,  179 
Phenylaminoacetic  acid,  fermentation  of, 

70 

Philothion,  66 
Phylogeny  of  yeasts,  139 
Physiological,  conditions  of  budding,  112 

conditions  of  sporulation,  114 

variations,  184 
Physiology  of  yeasts,  53 


Pichia  alcoholophila,  281 

californica,  278 

calliphora,  281 

farinosa,  279 

general  characteristics  of,  195 

hyalospora,  277 

mandshuricus,  282 

membranaefaciens,  276 

membranaefaciens,  I  and  II,  277 

orientalia,  283 

polymorpha,  281 

radaisii,  279 

suavebleus,  280 

tamarindorum,  278 

tauricus,  278 

Pickle  brine,  yeast  from,  305 
Picroformol  solution,  160 
Pineapples,  yeast  from,  337 
Plate  cultures,  173 
Polymorphism,  178 
Potassium  in  yeast  nutrition,  68 
Preparation  of,  Buchner's  zymase,  90 

yeast  juice,  58 
Preservation  of  yeasts,  164 
Pressure,  effect  on  yeasts,  111 
Prevalence  of  alcoholic  fermentation,  84 
Products  of  fermentation-,  89 
Properties  of  Buchner's  zymase,  89 
Proteolytic  enzymes,  59 
Prunase,  66 
Pseudomonilia,  albomarginata,  378 

cartilaginosa,  379 

mesenterica,  379 

rubescens,  379 
Pseudosaccharomyces,  africanus,  325 

antillarum,  327 

apiculatus,  323 

apiculatus  parasiticus,  324 

austricus,  325 

cortici,  325 

general  characteristics,  196 

Germanii,  326 

indicus,  327 

Jensenii,  326 

Lafari,  326 

Lindneri,  326 

Malaianus,  326 

Mulleri,  325 

of  Will,  327 

santranzensis,  327 

Stevensi,  318 

Willii,  326 


422 


INDEX  OF  SUBJECTS 


Ptyelus  lineatus,  yeasts  in,  127 
Pulque,  yeast,  257 
yeast  from,  282 

Pure  cultures,  Lindner's  method,  157 
Purpose  of  glycogen  in  yeasts,  76 
Pyroracemic  acid,  fermentation  of,  64 

RADIOACTIVE     emanations,     effect     on 

yeasts,  109 
Raffinase,  62 
Raulin's  medium,  149 
Red  Torula  No.  36,  315 
Reducing  enzymes,  66 
Reduction    of    sulfur    compounds    by 

yeasts,  67 
Relation  of,  glycogen  to  fermentation, 

77,  78 

yeasts  to  oxygen  concentration,  82 
Relationships  of  the  yeasts,  139 
Rennin  of  yeast,  60 

Resistance  of  yeasts  to  sun  and  light,  135 
Respiration  in  yeasts,  80 
Retardation  of  sporulation,  116 
Retrogradation  of  copulation,  25 
Roger's  torula,  313 
Role  of  chromatin,  42 
Rose's  Torula,  305 

SAAZ,  AND  FROHBERG  yeast,  233 

yeast,  233 
Saccharomyces,  acidi  lactici,  265 

anamensis,  268 

anginal,  270 

aquifolii,  246 

awamori,  302 

Bailii,  ameboid  forms  in,  27 

batatae,  248 

bayanus,  246 

Behrensianus,  229 

Blanchardii,  271 

brassicae,  303 

carlsbergensis,  234 

cartilaginosus,  248 

cerevisiae,  231 

cerevisiae  cells,  7 

cerevisiae,  effect  of  pressure  on,  111 

cerevisiae,  germination  of  ascospores 
in,  29,  35,  52 

chevalieri,  262 

comesii,  230 

conglomeratus,  269 

conomeli  limbati,  360 


Saccharomyces,  coreanus,  255 

ellipsoideus,  241 

Etienne,  263 

exiguus,  254 

family  of,  193 

flava  lactis,  265 

fragilis,  264 

from  Shiro-Koji,  251 

general  characteristics,  195 

granulatus,  271 

guttulatus,  230 

guttulatus,  asc  formation,  34 

Hansenii,  269 

ilicis,  246 

intermedius,  238 

japonicus,  322 

Jorgensenii,  256 

keiskeana,  322 

lactis,  0  and  a,  266 

lactis,  7,  226 

lebenis,  310 

lemonnieri,  273 

Lindnerii,  260 

Ludwigii,  227 

Ludwigii,  abnormal  germination  of 
ascospores,  35 

Ludwigii,  ascospore  formation,  50 

Ludwigii,  germination  of  ascospores 
in,  23,  24,  30,  31 

Ludwigii,  mycelial  formations  in,  9 

Ludwigii,  parthenogenetic  variety,  28 

macropsidis  lanionis,  325 

mali  Duclauxi,  264 

mali  risleri,  249 

mandshuricus,  253 

Mangini,  261 

marxianus,  252 

membranogenes,  357 

minor,  272 

monacensis,  235 

multisporus,  248 

mycoderma,  336 

mycoderma  punctisporus,  233 

nokkoensis  characterization,  204 

octosporus,  copulation  and  asc  forma- 
tion, 17 

olei,  313 

paradoxus,  260 

pastorianus,  237 

piriformis,  247 

rouxii,  256 

sake,  247 


INDEX  OF  SUBJECTS 


423 


Saccharomyces,  spec,  301 

T  and  V  of  Rose,  252 

taette,  268 

theobromae,  269 

Tokyo,  250 

tumefaciens,  270 

turbidans,  244 

tyrocola,  307 

unisporus,  264 

uvarum,  272 

validus,  240 

vini  Muntzii,  243 

vordermannii,  247 

willianus,  245 

Yeddo,  257 

Zopfii,  254 

Saccharomycodes,    general    characteris- 
tics, 194 

Saccharomycopsis,    general   characteris- 
tics, 194 

Saito's  Mycoderma,  335 
Sake,  yeast  from,  247 
Salt,  yeast  from,  270 

yeast  in,  306 
Sarcoma,  yeast  from,  351 
Sauerkraut,  yeast,  336 
Schizosaccharomyces,  aphalarae  calthae, 
characterization,  202 

asporus,  characterization,  202 

chermetis  abietis,  characterization,  205 

formosensis,  characterization,  203 

general  characteristics,  193,  197 

mellacei,  characterization,  200 

octosporus,  abnormal  germination  of 
ascospores,  36 

octosporus,  characterization,  197 

Pombe,  characterization,  199 

Pombe,  copulation  and  asc  formation, 
18 

Pombe,  germination  of  ascospores,  35 

sautawensis,  characterization,  204 
Schwanniomyces,  general  characteristics, 
194 

occidentalis,  224 

occidentalis,  formation  of  ascs  in,  26 
Schwanniomyces  occidentalis,   germina- 
tion of  ascospores,  34 
Scum  formation,  113 

temperature  limits,  171 
Sexuality,  15,  170 
Shape,  and  dimensions,  167 

of  yeast  cells,  6 


Shapes  of  ascospores,  15 

"Shoju"  yeast,  215 

Similarity  of  sporangia  to  ascs,  2 

Size  of  yeast  cells,  6 

Slater's  method  for  studying  yeasts,  158 

Soil,  yeast,  281,  302 

yeast,  from,  287,  288 

yeasts  in,  135,  221 
Source  of,  carbon  for  yeasts,  75 

yeasts,  134 

Sources  of  oxygen,  80,  81 
"Soya"  mash,  yeasts  in,  330 

yeasts  in,  209 
Spores,  detection  of,  159 
Sporidia  of  Ustilaginales,  1 
Sporulation,  13 

cytological  changes  during,  48 

Gorodkowa's  medium  for,  152 

methods  of  securing,  151 

physiological  conditions,  114 
Sputum,  yeast  from,  355 
Stab  cultures,  173 
Staining  ascospores,  52 
Staining  of  nucleus,  160 
Streak  cultures,  173 
Streptococcus,  b,  in  Kefir,  124 

lebenis,  126 

Structure,  of  yeast  nucleic  acid,  57 
Sucrase,  63 

Sugar,  enzymes  for,  61 
.  raw,  yeast  in,  309 

yeasts  from,  204 
Sulfur  in  yeast  nutrition,  68 
Symbiosis,  in  yeasts,  124 

of  yeast  and  bacterium,  116 

TEMPERATURE,  108 

influence  on  sporulation,  117 

of  ascospore  formation,  237 

of  scum  formation,  238 
Test  for  vitamines,  130 
Thermal  death  points,  108,  168 
Thiocyanates  as  sources  of  carbon,  sul- 
fur and  nitrogen,  71 
Thrush,  yeasts  from,  361 
Tivy,  yeast  from,  279 
Tomato,  yeast  on,  291 
Top  yeast,  231 

transformation  into  bottom  yeasts,  186 
Torula,  amara,  311 

bogoriensis  rubra,  323 

brettanomyces,  299 


424 


INDEX  OF  SUBJECTS 


Torula,  cinnabarina,  315 

colliculosa,  301 

general  characteristics,  196 

communis,  309 

glutinis,  316 

of  Hansen,  295 

Holmii,  304 

Kephir,  307,  311 

lactis,  308 

of  Lindner  and  Meissner,  299 

methods  of  characterization,  176 

mucilaginosa,  314 

nigra,  328 

novae  carlsbergiae,  299 

of  Pearse  and  Barker,  302 

pulcherrima,  314 

of  Rose,  305 

rubefaciens,  323 

"Soya"  mash,  330 

sp.  306 

thermantitonum,  304 

vaccine  pulp,  304 

Will's,  295 
Torulaspora,  general  characteristics,  194 

Delbriicki,  225 
Toxins  in  yeast,  67 
Transformations    of    bottom    into    top 

yeasts,  186 

Transportation  of  yeasts  through  air,  136 
Transverse  division  of  yeasts,  10,  48 
Trehalase,  63 
True  yeasts,  3 

ULTRAVIOLET  light  and  yeasts,  109 
Urea  in  yeast  nutrition,  71 
Utilization  of,  maltose,  76 
of  nitrates,  70 

VACCINE  pulp,  yeast  from,  304 
Vacuole  nucleare,  37 
Variations,  morphological,  179 

physiological,  184 
Varieties  of  Willia  anomala,  284 
Vitamines  in  yeasts,  129 

WEHMER'S  yeast,  305 

Will's  yeasts,  233 

Willia,  anomala,  284,  287 

anomala,  germination  of  ascospores,  33 
anomala,  varieties  of,  284,  285,  286 
belgica,  287 


Willia,  general  characteristics,  195 
Saturnus,  288 
Saturnus,  germination  of  ascospores, 

33 

Saturnus,  isogamic  copulation,  33 
wichmanni,  287 
Warm  stage,  158 
William's  test  for  vitamines,  130 
Will's  Torula,  295 
Wohl's  theory  of  alcoholic  fermentation, 

95 

Wortmann  and  Delbriick's  theory  of  fer- 
mentation, 100 

YEAST,  from  dough,  248 

"  Yeast  F"  of  Rose,  27,  219 

Yeast  forms,  132 

Yeast  G,  220 

Yeast  G  of  Pearse  and  Barker,  21 

Yeasts,  historical,  131 

Yeasts  in  cancer,  121 

Yeast  juice,  preparation  of,  58 

Yeast  cells  from  other  fungi,  1 

Yeast  membrane,  45 

Yeast  treatment  of  furunculosis,  129 

ZYGOSACCHAROMYCES,  BAILII,  character- 
istics, 211 

Bailii  cells,  211 

Barkerii,  206 

bisporus,  220 

chevalieri,  216 

general  characteristics,  194 

japonicus,  207 

javanicus,  207 

lactis  a,  210   ' 

major,  215 

mandshuricus,  212 

mellis  acidi,  212 

Nadsonii,  213 

Pastori,  heterogamic  copulation,  22 

prioranus,  206 

prioranus,  copulation  and  asc  forma- 
tion, 20 

salsus,  218 

soja,  258 

soya,  209 
Zygospore,  31 
Zymase,  89 
Zymine,  90 


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14  DAY 

MAR     2 199 

mrrunNii 


28  DAY 

SEP  0  3  1996 
SEP  2  0 1996 


28 


APR  2  8  1984    NOV  0  7  1996 

14 

OCT281996 


JUN  -  8  19.94 


10m-l,'57(C4267s4)4128 


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3   1378  00611   4097 


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